15-thia steroid compounds and compositions

ABSTRACT

The invention relates to the use of compounds to treat a number of conditions, such as thrombocytopenia, neutropenia or the delayed effects of radiation therapy. Compounds that can be used in the invention include methyl-2,3,4-trihydroxy-1-O-(7,17-dioxoandrost-5-ene-3β-yl)-β-D-glucopyranosiduronate, 16α,3α-dihydroxy-5α-androstan-17-one or 3,7,16,17-tetrahydroxyandrost-5-ene, 3,7,16,17-tetrahydroxyandrost-4-ene, 3,7,16,17-tetrahydroxyandrost-1-ene or 3,7,16,17-tetrahydroxyandrostane that can be used in the treatment method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional application is a continuation of pending U.S.application Ser. No. 12/633,721, filed Dec. 8, 2009, which is acontinuation of U.S. application Ser. No. 11/234,675, filed Sep. 23,2005, now abandoned, which is a continuation application of U.S.application Ser. No. 10/087,929, filed Mar. 1, 2002, now abandoned, eachof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods to make or use compounds, such as16α-bromo-3β-hydroxy-5α-androstane-17-one (16α-bromoepiandrosterone orhereafter “BrEA”) 3,7,16,17-tetrahydroxyandrost-5-ene,3,7,16,17-tetrahydroxyandrostane, 3,17-dihydroxy-16-haloandrostane,methyl2,3,4-trihydroxy-1-O-(7,17-dioxoandrost-5-ene-3β-yl)-β-D-glucopyranosiduronateand related compounds. The invention relates to the use of compounds totreat a number of conditions, such as thrombocytopenia and neutropenia.

DESCRIPTION OF THE INVENTION

Methods to prepare dehydroepiandrosterone (“DHEA”) and other steroidsand their biological properties have been described, see, e.g., U.S.Pat. Nos. 2,833,793, 2,911,418, 3,148,198, 3,471,480, 3,976,691,4,268,441, 4,427,649, 4,542,129, 4,666,898, 4,956,355, 5,001,119,5,043,165, 5,077,284, 5,028,631, 5,110,810, 5,157,031, 5,162,198,5,175,154, 5,277,907, 5,292,730, 5,296,481, 5,372,996, 5,387,583,5,407,684, 5,424,463, 5,461,042, 5,478,566, 5,506,223, 5,518,725,5,527,788, 5,527,789, 5,532,230, 5,559,107, 5,562,910, 5,583,126,5,585,371, 5,587,369, 5,591,736, 5,593,981, 5,610,150, 5,635,496,5,641,766, 5,641,768, 5,656,621, 5,660,835, 5,686,438, 5,696,106,5,700,793, 5,707,983, 5,709,878, 5,710,143, 5,714,481, 5,728,688,5,736,537, 5,744,462, 5,753,237, 5,756,482, 5,776,921, 5,776,923,5,780,460, 5,795,880, 5,798,347, 5,798,348, 5,804,576, 5,807,848,5,807,849, 5,811,418, 5,824,313, 5,824,668, 5,824,671, 5,827,841,5,837,269, 5,837,700, 5,843,932, 5,846,963, 5,859,000, 5,872,114 and5,872,147; German patent numbers 2035738 and 2705917; PCT publicationnumbers WO 95/21617, WO 97/48367, WO 98/05338, WO 98/50040, WO 98/50041,WO 98/58650; European publication number 0020029; E. R. Glazier, J. Org.Chem. 1962 27:2937-2938, Ben-David, et al., Proc. Soc. Expt. Biol. Med.1967 125:1136-1140, Coleman et al., Diabetes 1982 31:830, Oertel, etal., J. Steroid Biochem. 1972 3:493-496, Pashko, et al., Carcinogenesis1981 2:717-721, Schwartz et al., Nutr. Cancer 1981 3:46-53; Dyner etal., J. Acquired Immune Deficiency Syndromes 1993 6:459-465; A. A.Afanasii and Y. A. Titov, Total Steroid Synthesis, Plenum Press, NewYork, 1970, see, e.g., p 1-304.

The use DHEA and other steroids in various applications have beendescribed, e.g., U.S. Pat. Nos. 5,869,090, 5,863,910, 5,856,340,5,824,668, 5,804,576, 5,753,237, 5,714,481, 5,709,878, 5,407,684,5,206,008, 5,077,284, 4,978,532, 4,898,694, 4,542,129, 3,711,606 and3,710,795. U.S. Pat. No. 4,956,355 and PCT publication number WO97/48367, have described the use of certain steroid compounds to treatcertain virus or bacterial infections, such as human immunodeficiencyvirus (“HIV”) infection.

Various biological effects and/or metabolic conversions of steroidcompounds have also been described, e.g., Batta et al., J. Biol. Chem.1986 25:127-133, Belli et al., Liver 1991 11:162-169, Bhattacharjee etal., Anal. Biochem. 1992 201:233-236, Blake et al., Int. J. PeptideProtein Res. 1982 20:97-101, 1986 25:127-133, Bonaventura, Am. J.Obstet. Gynecol. 1978 131:403-409, Bucala et al., J. Steroid Biochem.1986 25:127-133, Carey et al., Biochem. 1981 20:3637-3648, Chen et al.,Carcinogenesis 1999 20:249-254, Chen et al., Carcinogenesis 199819:2187-2193, Chow et al., Antisense Res. Dev. 1994 4:81-86, Citro etal., Dis. Colon Rectum 1994 37(2 Suppl):5127-5132, Cleary, Proc. Soc.Exp. Biol. Med. 1991 196:8-16, Cleary, Int. J. Biochem. 1990 22:205-210,Crawford et al., Lab. Invest. 1994 71:42-51, Danenberg et al.,Antimicrob. Agents Chemother. 1992 36:2275-2279, Dotzlaw et al., CancerRes. 1999 59:529-532, Falany et al., J. Steroid Biochem. Mol. Biol. 199448:369-375, Faredin et al., J. Investigative Dermatol. 1969 52:357-361,Galigniana et al., Mol. Pharmacol. 1999 55:317-323, Goto et al., J.Chromatogr. 1983 276:289-300, Grenot Biochem. 1992 31:7609-7621,Hofbauer et al., Life Sci. 1999 64:671-679, Huijghebaert et al., J.Lipid Res. 1986 27:742-752, Hurd et al., Oncogene 1999 18:1067-1072,Iida et al., J. Lipid Res. 1995 36:628-638, Jellinck et al., Steroids1967 10:329-346, Jonsson et al., J. Pediatr. Gastroenterol. Nutr. 199520:394-402, Kalimi et al, Mol. Cell. Biochem. 1994 131:99-108, Kramer etal., J. Biol. Chem. 1994 269:10621-10627, LaRochelle et al., Steroids1984 43: 209-217, Liao et al., Carcinogenesis 1998 19:2173-2180,Lillienau et al., J. Clin. Invest. 1992 89:420-431, Loria,Psychoneuroendocrinology 1997 22:S103-S108, Luscher et al Mol. Immunol.1983 20:1099-1105, Manna et al., J. Biol. Chem. 1999 274:5909-5918,Marschall et al., J. Biol. Chem. 1989 264:12989-12993, Medh et al.,Cancer Res. 1998 15:3684-3693, Mohan et al., Steroids 1992 57:244-247,Munoz de Toro et al., J. Steroid Biochem. Mol. Biol. 1998 67:333-339,Padgett et al., J. Neuroimmunol. 1998 84:61, Padgett et al., Ann. N.Y.Acad. Sci. 1995 774:323, Padgett et al., J. Immunol. 1994 153:1544-1552,Pashko et al., Carcinogenesis 1984 5:463-466, Pashko et al.,Carcinogenesis 1981 2:717, Petrylak et al., J. Clin. Oncology 199917:958-967, Podesta et al., Steroids 1996 61:622-626, Regelson et al.,Ann. N.Y. Acad. Sci. 1994 719:564, Schmassmann et al., Gastroenterology1993 104:1171-1181, Schmassmann et al., Hepatology 1990 11:989-996,Schreiber et al., Lancet 353:459-461, Schreiber, Neth. J. Med. 199853:S24-31, Schwartz et al., Cancer Res. 1988 48:4817, Shahidi et al.,Biochem. Biophys. Res. Commun. 1999 254:559-565, Steer et al., Ann.Rheum. Dis. 1998 57:732-737, Suzuki et al., Steroids 1998 63:672-677,Suzuki et al., Steroids 1996 61:296-301, Swaan et al., BioconjugateChem. 1997 8:520-525, Tang et al, Anticancer Drug Res. 1998 13:815-824,Thomas et al., J. Steroid Biochem. 1986 25:103-108, Utsumi et al.,Cancer Res. 1999 59:377-381, Vanden Heuvel, J. Nutr. 1999 129(2SSuppl.):5755-5805, Wang et al., Endocrinology 1998 139:3903-3912, Wonget al., J. Biol. Chem. 1999 274:5443-5453, Xie et al., Endocrinology1999 140:219-227, Yen et al., Lipids 1977 12:409-413, Zackheim et al.,Arch. Dermatology 1998 134:949-954, Zhang et al., Biochim. Biophys. Acta1991 1096:179-186, Zhu et al., Carcinogenesis 1988 19:2101-2106.

Mammalian immune responses to infections or other conditions are oftencharacterized by responses mediated by different effector cellpopulations. In some situations, helper T cells designated Th1 in themurine system, facilitate immune effector functions that are typicallydominated by cell-mediated responses. In other cases, helper T cellsdesignated Th2 cells facilitate immune effector functions that aretypically dominated by humoral responses. A vigorous Th1 response isusually desirable to help clear infections or to slow the progression ofan infection. When a subject's immune response is biased to, ordominated by, a Th2-type response, the cytokines associated with the Th2response tend to suppress the immune system's capacity to mount avigorous Th1 response at the same time. The converse is also generallytrue. When mammalian immune responses begin to result in an increasingTh2 response, the Th1 response to the same condition tends to weaken.Insufficient Th1 responses may be associated with progression of someinfections or other conditions, see, e.g., M. Clerici and G. M. Shearer,Immunol. Today 14:107-111, 1993; M. Clerici and G. M. Shearer, Immunol.Today 15:575-581, 1994. The invention provides compounds andcompositions useful to enhance Th1 immune responses.

Hemopoiesis or hematopoiesis is the formation and development of thevarious types of blood cells and their progenitor cells. Mature cellsare found in circulation or tissues such as the lymph nodes or thethymus. Many of the stem cells that give rise to mature forms reside inthe bone marrow, although some may circulate in the blood for some time.Clinical blood cell deficiencies such as thrombocytopenia, neutropeniaor erythropenia can arise from causes such as impaired hemopoiesis orabnormal loss or destruction of mature or immature blood cells.

Thrombocytopenia (“TP”), abnormally low platelet counts, can arise fromimpaired platelet production, sequestration of platelets in the spleenor abnormal loss of circulating platelets. Impaired production canresult from causes such as chemotherapies or radiation therapies.Abnormal loss of circulating platelets is often associated withautoreactive antibodies that bind to platelets and reduce their lifespan. These underlying causes give rise to the various clinical forms ofTP, such as autoimmune neonatal TP, immune thrombocytopenic purpra,radiation induced TP, chemotherapy induced TP and amegakaryocitic TP.

A number of treatment options are available and the selection of atreatment typically depends on the source of the disorder and itsseverity. Treatments include administering one or more of glucocorticoidsteroids (e.g., prednisone, prednisolone), human IgG antibodies,anti-Rh(D)⁺ antibodies for Rh (D)⁺ patients, an androgen such asdanazol, vinca alkaloids (e.g., vincristine, vinblastine),thrombopoietin and immunosuppresants (e.g., azathioprine,cyclophosphamide). Splenectomy is also indicated, for example when firstline treatments fail. The goal of treatment is typically to increaseplatelet counts to 20,000 mm⁻³ or more typically to at least about50,000 mm⁻³ and to maintain these levels.

Although the treatment options to increase platelet levels are generallyknown, they usually have a number of drawbacks. For example, infusion ofIgG antibodies is not always effective and the treatment is relativelyexpensive. Other treatments, such as prednisone are also not alwayseffective and they typically are discontinued or tapered off afterseveral weeks due to toxicity or unwanted side effects. Splenectomy,which is relatively expensive and invasive, is also not alwayseffective.

The sources of thrombocytopenia and treatment options have beendescribed. See, e.g., Hematology—Basic Principles and Practice, 3^(rd)edition, R. Hoffman, E. J. Benz Jr. et al., editors, ChurchillLivingstone, New York, 2000 (see, e.g., Chapters 126-129 and 131 atpages 2096-2154 and 2172-2186), PCT publication WO 200035466.

Neutropenia (“NP”), is considered to exist clinically when neutrophilsdrop to below a level considered normal. NP can arise from impairedproduction of neutrophil precursors or mature neutrophils, movement ofneutrophils from the circulation to tissue, abnormal circulatingneutrophil loss or a combination of these causes. Impaired neutrophilproduction can be acquired from, e.g., treatment with a cytotoxic orcytostatic drug, chemotherapy, radiation therapy or an autoimmuneresponse. The abnormal loss of circulating neutrophils in autoimmunityis associated with autoreactive antibodies that bind to the cells andreduce their life span. These underlying causes give rise to the variousclinical forms of NP, such as postinfectious NP, drug-induced NP,autoimmune NP, or chronic idiopathic NP.

The sources of NP and treatment options have been described. See, e.g.,Hematology—Basic Principles and Practice, 3^(rd) edition, R. Hoffman, E.J. Benz Jr. et al., editors, Churchill Livingstone, New York, 2000 (see,e.g., Chapters 19, 41, 51, 79, 134 and 137 at pages 297-331, 720-762,939-979, 1443-1500, 2220-2248 and 2257-2263).

The use of 3β-hydroxyandrost-5-ene-17-one, 3β,17β-dihydroxyandrost-5-eneand other steroids to modulate immune functions or to stimulatemyelopoiesis has been described, see, e.g., M. H. Whitnall et al., Intl.J. Immunopharmacology 2000 22:1-14. U.S. Pat. Nos. 5,162,198, 5,206,008,5,292,730, 5,407,684, 5,461,042, 5,461,768, 5,478,566, 5,585,371,5,635,496, 5,641,766, 5,753,237, 5,837,269, 5,885,977 and 5,919,465, PCTpublication nos. WO93/20696 and WO99/25333.1. Porsova-Dutoit et al.,Physiological Res. 2000 49 (Suppl. 1):543-556, R. L. Jesse et al., Ann.N.Y. Acad. Sci. 1995 774:281-290 and U.S. Pat. Nos. 5,532,230, 5,811,418and 5,846,963 discuss the capacity of 3β-hydroxyandrost-5-ene-17-one,its 3-sulfate derivative and other steroids to affect platelet andneutrophil aggregation or their adhesion to endothelial cells.

U.S. Pat. Nos. 4,908,358 and 4,902,681 describe the capacity ofcompounds such as 5α-pregnan-3,20-dione, cortexolone,17-hydroxyprogesterone and 16α-methylprogesterone to inhibit theclearance of antibody-coated cells from circulation in disorders such asimmune thrombocytopenic purpura or immune hemolytic anemia.

U.S. Pat. Nos. 5,532,230, 5,686,438, 5,753,640 and 5,811,418 and J.Bratt and M. Heimburger, Scand. J. Rheumatol. 1999 28:308-313 describethe capacity of compounds such as 3β,7β-dihydroxyandrost-5-ene-17-one,prednisolone, and 3β-hydroxyandrost-5-ene-17-one to limit tissue damagein ischemic tissues by inhibiting adhesion of cells such as neutrophilsto endothelial cells or to treat pulmonary hypertension.

U.S. Pat. No. 5,859,000 describes the capacity of compounds such as3β,7β-dihydroxyandrost-5-ene-17-one and 3β-hydroxyandrost-5-ene-17-oneto reduce mast cell mediated allergic reactions.

U.S. Pat. No. 5,763,433 and PCT publication WO 96/35428 describe thecapacity of compounds related to dehydroepiandrosterone and16α-halodehydroepiandrosterone to modulate immune responses and to treatconditions certain immune related conditions such as systemic lupuserythematosus.

U.S. Pat. Nos. 5,925,630, 5,939,545 and 5,962,443 describe the capacityof 19-nur-pregnane steroids, 3α-hydroxy-5α-pregnan-20-one and relatedsteroids to modulate certain neurological activities such ashypothalamic function and GABA receptor activity.

Some proteins such as interleukin-6 (“IL-6”), erythropoietin (“EPO”) andthrombopoietin (“TPO”) have been examined for their capacity to enhancevarious aspects hemopoiesis, e.g., Hematology—Basic Principles andPractice, 3^(rd) edition, R. Hoffman, E. J. Benz Jr. et al., editors,Churchill Livingstone, New York, 2000 (see, e.g., Chapter 14 at pages154-202), O. J. Borge et al., Blood 1996 88:2859-2870, M. Cremer et al.,Ann. Hematol. 1999 78:401-407, Y. Sasaki et al., Blood 199994:1952-1960, U.S. Pat. No. 5,879,673. Recombinant IL-6 was shown inmodel systems to affect platelet counts in peripheral circulation, e.g.,Stahl et al., Blood 1991 78:1467-1475, although significant toxicitiesare associated with its administration to humans, e.g., Andus et al.,FEBS Lett. 1987 221:18, J. Gauldie et al., P.N.A.S. U.S.A. 198784:7251-7255, T. Geiger et al., Eur. J. Immunol. 1988 18:717-721. TheIL-6 molecule has been described in detail, e.g., publication no. WO88/00206. Administration of proteins is typically expensive, givenfactors such as the complexity of producing pharmaceutical gradematerial.

The capacity of various compounds or agents such as deuterium oxide,lithium and butyrate to affect or to participate in biological functionsof cells such as neutrophils has been described. See, e.g., M. F. Tsanand R. M. Turkall, Inflammation 1982 6:387-396, M. Nakamura et al., Exp.Cell Res. 1976 102:429-431, P. Blier et al., Int. Clin. Psychopharmacol.1998 13:137-140, N. Turkozkan et al., Int. J. Biochem. 199325:1501-1504, L. V. Deriy et al., Biochem. Biophys. Res. Commun. 2000275:241-246, M. T. Elghetany et al., Leuk. Res. 1997 21:801-806, E.Brandt et al., J. Leukocyte Biol. 2000 68:125-130, M. Boussac and J.Garin, Electrophoresis 2000 21:665-672, M. Niwa et al., Life Sci. 200018:1525-1534, D. A. Moulding et al., J. Leukocyte Biol. 1999 65:875-882and D. Moulding et al., Biologicals 1996 24:301-306.

There is a current need for cost-effective pharmaceutical agents ortreatment methods that are more effective in treating deficiencies ofblood cells or reducing their symptoms. The present invention providestherapeutic agents and treatment methods to treat hemopoiesisdeficiencies and disorders such as TP and NP. The agents and methods arethus useful to reduce one or more symptoms associated with any of theseconditions. Also, the use of the invention agents and methods can becombined with one or more conventional treatments for these disorders.

Summary of invention embodiments. The invention provides a method totreat a blood cell deficiency in a subject in need thereof comprisingadministering to the subject, or delivering to the subject's tissues, aneffective amount of a compound of formula 1

wherein, each R¹, R², R³, R⁴, R⁵, R⁶ and R¹⁰ independently are —H,—OR^(PR), —SR^(PR), —N(R^(PR))₂, —O—Si—(R¹³)₃, —CHO, —CHS, —CH═NH, —CN,—SCN, —NO₂, —OSO₃H, —OPO₃H, an ester, a thioester, a thionoester, aphosphoester, a phosphothioester, a phosphonoester, a phosphiniester, asulfite ester, a sulfate ester, an amide, an amino acid, a peptide, anether, a thioether, an acyl group, a thioacyl group, a carbonate, acarbamate, a halogen, an optionally substituted alkyl group, anoptionally substituted alkenyl group, an optionally substituted alkynylgroup, an optionally substituted aryl moiety, an optionally substitutedheteroaryl moiety, an optionally substituted heterocycle, an optionallysubstituted monosaccharide, an optionally substituted oligosaccharide, anucleoside, a nucleotide, an oligonucleotide, a polymer, or,

one or more of both R¹, R², R³ or R⁴ together comprise an independentlyselected spiro ring, or

one more of R¹, R², R³, R⁴, R⁵, R⁶ and Fr are ═O, ═S, ═N—OH, ═CH₂, or aspiro ring and the hydrogen atom or the second variable group that isbonded to the same carbon atom is absent, or,

one or more of two adjacent R¹-R⁶ and R¹⁰ comprise an independentlyselected an acetal, a thioacetal, ketal or thioketal, or

all R³ and R⁴ together comprise a structure of formula 2

R⁷ is —C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—C(R¹⁰)₂—,—C(R¹⁰)₂—O—C(R¹⁰)₂—, —C(R¹⁰)₂—S—C(R¹⁰)₂—, —C(R¹⁰)₂—NR^(PR)—C(R¹⁰)₂—,—O—, —O—C(R¹⁰)₂—, —S—, —S—C(R¹⁰)₂—, —NR^(PR)— or —NR^(PR)—C(R¹⁰)₂—;

R⁸ and R⁹ independently are —C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—, —O—,—O—C(R¹⁰)₂—, —S—, —S—C(R¹⁰)₂—, —NR^(PR)— or —NR^(PR)—C(R¹⁰)₂—, or one orboth of R⁸ or R⁹ independently are absent, leaving a 5-membered ring;

R¹³ independently is C₁₋₆ alkyl;

R^(PR) independently is —H or a protecting group;

D is a heterocycle or a 4-, 5-, 6- or 7-membered ring that comprisessaturated carbon atoms, wherein 1, 2 or 3 ring carbon atoms of the 4-,5-, 6- or 7-membered ring are optionally independently substituted with—O—, —S— or —NR^(PR)— or where 1, 2 or 3 hydrogen atoms of theheterocycle or where 1, 2 or 3 hydrogen atoms of the 4-, 5-, 6- or7-membered ring are substituted with —OR^(PR), —SR^(PR), —N(R^(PR))₂,—O—Si—(R¹³)₃, —CHO, —CHS, —CH═NH, —CN, —SCN, —NO₂, —OSO₃H, —OPO₃H, anester, a thioester, a thionoester, a phosphoester, a phosphothioester, aphosphiniester, a sulfite ester, a sulfate ester, an amide, an aminoacid, a peptide, an ether, a thioether, an acyl group, a thioacyl group,a carbonate, a carbamate, a halogen, an optionally substituted alkylgroup, an optionally substituted alkenyl group, an optionallysubstituted alkynyl group, an optionally substituted aryl moiety, anoptionally substituted heteroaryl moiety, an optionally substitutedheterocycle, an optionally substituted monosaccharide, an optionallysubstituted oligosaccharide, a nucleoside, a nucleotide, anoligonucleotide or a polymer, or,

one more of the ring carbons in D are substituted with ═O, ═S, ═N—OH,═CH₂, or a spiro ring, or

one or more of two adjacent ring carbons in D comprise an independentlyselected acetal, thioacetal, ketal or thioketal, or

D comprises two 5- or 6-membered rings, wherein the rings are fused orare linked by 1 or 2 bonds, or a metabolic precursor or a biologicallyactive metabolite thereof. In some embodiments, the compound is not5-androstene-3β-17-one, 5-androstene-3β,17β-diol,5-androstene-3β,7β,17β-triol or a derivative of any of these threecompounds that can convert to these compounds by hydrolysis.

Invention embodiments also provide a method to modulate an immune orcellular response in a subject in need thereof comprising administeringto the subject, or delivering to the subject's tissues, an effectiveamount of a compound of formula 1

wherein,

each R¹, R², R³, R⁴, R⁵, R⁶ and R¹⁰ independently are —H, —OR^(PR),—SR^(PR), —N(R^(PR))_(2i)—O—Si—(R¹³)₃, —CHO, —CHS, —CH═NH, —CN, —SCN,—NO₂, —OSO₃H, —OPO₃H, an ester, a thioester, a thionoester, aphosphoester, a phosphothioester, a phosphonoester, a phosphiniester, asulfite ester, a sulfate ester, an amide, an amino acid, a peptide, anether, a thioether, an acyl group, a thioacyl group, a carbonate, acarbamate, a halogen, an optionally substituted alkyl group, anoptionally substituted alkenyl group, an optionally substituted alkynylgroup, an optionally substituted aryl moiety, an optionally substitutedheteroaryl moiety, an optionally substituted heterocycle, an optionallysubstituted monosaccharide, an optionally substituted oligosaccharide, anucleoside, a nucleotide, an oligonucleotide, a polymer, or,

one or more of both R¹, R², R³ or R⁴ together comprise an independentlyselected spiro ring, or

one more of R¹, R², R³, R⁴, R⁵, R⁶ and Fr independently are ═O, ═S,═N—OH, ═CH₂, or a spiro ring, and the hydrogen atom or the secondvariable group that is bonded to the same carbon atom is absent, or,

one or more of two adjacent R¹-R⁶ and R¹⁰ comprise an independentlyselected acetal, thioacetal, ketal or thioketal;

all R³ and R⁴ together comprise a structure of formula 2

R⁷ is —C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—C(R¹⁰)₂—,—C(R¹⁰)₂—O—C(R¹⁰)₂—, —C(R¹⁰)₂—S—C(R¹⁰)₂—, —C(R¹⁰)₂—NR^(PR)—C(R¹⁰)₂—,—O—, —O—C(R¹⁰)₂—, —S—, —S—C(R¹⁰)₂—, —NR^(PR)— or —NR^(PR)—C(R¹⁰)₂—;

R⁸ and R⁹ independently are —C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—, —O—,—O—C(R¹⁰)₂—, —S—, —S—C(R¹⁰)₂—, —NR^(PR)— or —NR^(PR)—C(R¹⁰)₂—, or one orboth of R⁸ or R⁹ independently are absent, leaving a 5-membered ring;

R¹³ independently is C₁₋₆ alkyl;

R^(PR) independently is —H or a protecting group;

D is a heterocycle or a 4-, 5-, 6- or 7-membered ring that comprisessaturated carbon atoms, wherein 1, 2 or 3 ring carbon atoms of the 4-,5-, 6- or 7-membered ring are optionally independently substituted with—O—, —S— or —NR^(PR)— or where 1, 2 or 3 hydrogen atoms of theheterocycle or where 1, 2 or 3 hydrogen atoms of the 4-, 5-, 6- or7-membered ring are independently substituted with —OR^(PR), —SR^(PR),—N(R^(PR))₂, —O—Si—(R¹³)₃, —CHO, —CHS, —CH═NH, —CN, —SCN, —NO₂, —OSO₃H,—OPO₃H, an ester, a thioester, a thionoester, a phosphoester, aphosphothioester, a phosphiniester, a sulfite ester, a sulfate ester, anamide, an amino acid, a peptide, an ether, a thioether, an acyl group, athioacyl group, a carbonate, a carbamate, a halogen, an optionallysubstituted alkyl group, an optionally substituted alkenyl group, anoptionally substituted alkynyl group, an optionally substituted arylmoiety, an optionally substituted heteroaryl moiety, an optionallysubstituted heterocycle, an optionally substituted monosaccharide, anoptionally substituted oligosaccharide, a nucleoside, a nucleotide, anoligonucleotide or a polymer, or,

one more of the D ring carbons are substituted with ═O, ═S, ═N—OH, ═CH₂,or a spiro ring, or

two adjacent D ring carbons comprise an independently selected acetal,thioacetal, ketal or thioketal moiety, or

D comprises two 5- or 6-membered rings, wherein the rings are fused orare linked by 1 or 2 bonds, or a metabolic precursor or a biologicallyactive metabolite thereof, optionally provided that if the subject is inneed of enhanced hemopoiesis, the compound is not5-androstene-3β-ol-17-one, 5-androstene-3β,17β-diol,5-androstene-3β,7β,17β-triol or a derivative of any of these threecompounds that can convert to these compounds by hydrolysis. Immune andcellular response modulation includes enhancing Th1 immune responses,reducing Th2 immune responses, reducing inflammation and enhancinghemopoiesis.

In other embodiments, the invention provides a compound of formula 1,wherein two or three of R⁷, R⁸ and R⁹ independently are not —CHR¹⁰—, or—C(R¹⁰)₂— and wherein the compound is optionally present in acomposition that comprises one or more excipients.

Other embodiments include a method to enhance the expression of one ormore cytokines or interleukins that facilitate Th1 or Tc1 immuneresponses in a subject or to reduce the expression of one or morecytokines or interleukins that facilitate Th2 or Tc2 immune response ina subject comprising administering to the subject an effective amount ofa formula 1 compound, whereby the subject's Th1 or Tc1 immune responseis enhanced to the subject's undesired Th2 or Tc2 immune response isreduced.

A further embodiment is a method to modulate a subject's innateimmunity, Th1 immune responses, Tc1 immune responses, Th2 immuneresponses, Tc2 immune responses, or inflammation comprisingadministering an effective amount of a formula 1 compound to a subjector delivering the formula 1 compound to the subject's tissues.

Other embodiments are as described elsewhere in the specificationincluding the numbered embodiments and the claims.

Definitions. As used herein and unless otherwise stated or implied bycontext, terms that are used herein have the meanings that are definedhere. The descriptions of embodiments and examples that are describedillustrate the invention and they are not intended to limit it in anyway. Unless otherwise contraindicated or implied, e.g., by includingmutually exclusive elements or options, in these definitions andthroughout this specification, the terms “a” and “an” mean one or moreand the term “or” means and/or.

An “invention formulation”, “formulation” or the like means acomposition that one can administer to a subject, e.g., human or animal,without further manipulations that change the ingredients or theingredient proportions that are present. Formulations are suitable forhuman or veterinary applications and would typically have expectedcharacteristics for the formulation, e.g., parenteral formulations forhuman use would usually be sterile.

An “invention composition”, “composition” or the like means acomposition, that is a formulation or that can be an intermediate onecan use to make the formulations, i.e., a change(s) in an ingredient(s)or its amount(s) may be needed to make a formulation. Compositions mayalso comprise other types of materials, e.g., reagents for assays orcells that are optionally contacted with a formula 1 compound ormixtures of compounds. Thus, invention compositions include compositionswhere further processing may be required before it is a formulation,e.g., mixing or addition of a desired amount of an ingredient.

Phrases such as “administration of a compound of formula 1”, “treatmentwith a formula 1 compound” or similar terms mean that the compound(s) isadministered to, or delivered to, the subject or to the subject'stissues by one or more suitable methods, e.g., by an oral, topical,parenteral, buccal or sublingual route.

Expressions such as “a formula 1 compound(s)”, “a formula 1 compound”and the like mean invention compositions or formulations where one ormore than one formula 1 compound is present, e.g., in a composition, oris used in the disclosed method, typically 1, 2, 3 or 4, usually 1. Anyreference to a “formula 1 compound”, “one or more compounds of formula1” or the like means that the formula 1 compound can have the formula 2structure or any other structure disclosed herein that is within thedefinition of formula 1 compounds.

Reference to subject matter “as disclosed herein” such as a “therapeutictreatment or agent as disclosed herein”, a “dosing protocol as disclosedherein” or a “clinical condition or symptom as disclosed herein” or thelike means a treatment, agent, protocol, condition, symptom or the likethat is described herein or in any reference that is cited herein.

An “excipient”, “carrier”, “pharmaceutically acceptable carrier” orsimilar terms mean one or more component(s) or ingredient(s) that isacceptable in the sense of being compatible with the other ingredientsof invention compositions or formulations and not overly deleterious tothe patient, animal, tissues or cells to which the formulation is to beadministered.

As used here, “excipients” include liquids, such as benzyl benzoate,cottonseed oil, N,N-dimethylacetamide, a C₂₋₁₂ alcohol (e.g., ethanol),glycerol, peanut oil, a polyethylene glycol (“PEG”), vitamin E,poppyseed oil, propylene glycol, safflower oil, sesame oil, soybean oiland vegetable oil. Any solid excipient may be a fine powder orgranulated. Excipients, as used herein may optionally exclude one ormore excipient, e.g., chloroform, dioxane, vegetable oil, DMSO, otherexcipients or any combination of these. Excipients include one or morecomponents typically used in the pharmaceutical formulation arts, e.g.,one, two or more of fillers, binders, disintegrants, dispersants,preservatives, glidants and lubricants. Exemplary excipients includepovidone, crospovidone, corn starch, carboxymethyl cellulose,hydroxypropyl methylcellulose, microcrystalline cellulose, gum arabic,polysorbate 80, butylparaben, propylparaben, methylparaben, BHA, EDTA,sodium lauryl sulfate, sodium chloride, potassium chloride, titaniumdioxide, magnesium stearate, castor oil, olive oil, vegetable oil,buffering agents such as sodium hydroxide, monobasic sodium phosphate,dibasic sodium phosphate, potassium hydroxide, monobasic potassiumphosphate, dibasic potassium phosphate, tribasic potassium phosphate,potassium carbonate, potassium bicarbonate, ammonium hydroxide, ammoniumchloride, saccharides such as mannitol, glucose, fructose, sucrose orlactose any of which may be compressible or any of which may be spraydried.

A “subject” means a human or animal. Usually the animal is a mammal orvertebrate such as a primate, rodent, lagomorph, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus or Pan. Rodents and lagomorphsinclude mice, rats, woodchucks, ferrets, rabbits and hamsters. Domesticand game animals include cows, horses, pigs, sheep, deer, bison,buffalo, mink, felines, e.g., domestic cat, canines, e.g., dog, wolf andfox, avian species, e.g., chicken, turkey, emu and ostrich, and fish,e.g., trout, catfish and salmon. Subject includes any subset of theforegoing, e.g., all of the above, but excluding one or more groups orspecies such as humans, primates or rodents. Other subsets of subjectsinclude subjects of a given species or group of species of varying ages,e.g., young humans, e.g., about 1 week of age to about 9 years of age,adolescent humans, e.g., about 10-19 years of age, adult humans, e.g.,about 20-100 years of age, and mature adult or elderly humans, e.g., atleast about 55 years of age, at least about 60 years of age, at leastabout 65 years of age or a range of ages such as about 60-100 years ofage. Thus, as used herein, prevention or treatment of a disease,condition or symptom may include or exclude any subset of subjects thatare grouped by age.

The terms “effective amount”, “effective dose” or the like mean anamount of a formula 1 compound that is sufficient to elicit a desiredresponse, e.g., restoration of normal immune responsiveness in animmunodeficient subject to which it is administered or to detectablemodulation or amelioration of an immune or cellular parameter or aclinical condition or symptom. An effective amount may be a single doseor two or more subdoses of a formula 1 compound administered in one day,or it may be administered as multiple doses over a period of time, e.g.,over 2 days to about 1 year.

References such as “a disease”, “a disorder”, “a condition”, “a symptom’or “a complication” or the like mean that one or more disease, disorder,condition, symptom or complication may be treated.

Terms such as “use”, “treat”, “treatment”, “address” or the like in thecontext of using the formula 1 compounds in the treatment methods orother methods disclosed herein mean that a formula 1 compound isadministered to a subject, delivered to the subject's tissues orcontacted with tissues, cells or cell free systems, e.g., as describedherein or a reference cited herein. Typically such use or treatmentresults in detectable improvement in or amelioration of the condition orsymptom being treated. Such amelioration may be transient, e.g., lastingfor at least a few, e.g., about 1 to 24, hours or days, e.g., about 1-,7days, or amelioration may be prolonged, e.g., lasting about 8 to about60 days or more, or it may be permanent. A treatment may slow theprogression or severity of a disease or symptom. A use or treatment mayresult in detectable modulation in a relevant immune parameter such asmodulation of the level or activity of a target effector or suppressorimmune cell population, interleukin, cytokine, chemokine, immunoglobulinor modulation of the level or activity of a relevant transcriptionfactor, enzyme or cell biological activity. A treatment with thecompounds may be used to prevent the onset of a disease, symptom orcomplication or to ameliorate a preexisting disease, condition, symptomor complication, or to facilitate elimination of a disease, condition,symptom or complication.

“Ameliorate”, “amelioration”, “improvement” or the like means adetectable improvement or a detectable change consistent withimprovement occurs in a subject or in at least a minority of subjects,e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a range about between anytwo of these values. Such improvement or change may be observed intreated subjects as compared to subjects not treated with a formula 1compound, where the untreated subjects have, or are subject todeveloping, the same or similar disease, condition, symptom or the like.Amelioration of a disease, condition, symptom or assay parameter may bedetermined subjectively or objectively, e.g., self assessment by asubject(s), by a clinician's assessment or by conducting an appropriateassay or measurement, including, e.g., a quality of life assessment, aslowed progression of a disease(s) or condition(s), a reduced severityof a disease(s) or condition(s), or a suitable assay(s) for the level oractivity(ies) of a biomolecule(s), cell(s) or by detection of cellmigration within a subject. Amelioration may be transient, prolonged orpermanent or it may be variable at relevant times during or after aformula 1 compound is administered to a subject or is used in an assayor other method described herein or a cited reference, e.g., withinabout 1 hour of the administration or use of a formula 1 compound toabout 3, 6, 9 months or more after a subject(s) has received a formula 1compound.

The “modulation” of, e.g., a symptom, level or biological activity of amolecule, replication of a pathogen, cellular response, cellularactivity or the like, means that the cell, level or activity, or thelike is detectably increased or decreased. Such increase or decrease maybe observed in treated subjects as compared to subjects not treated witha formula 1 compound, where the untreated subjects have, or are subjectto developing, the same or similar disease, condition, symptom or thelike. Such increases or decreases may be at least about 2%, 5%, 10%,15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more or about withinany range about between any two of these values. Modulation may bedetermined subjectively or objectively, e.g., by the subject's selfassessment, by a clinician's assessment or by conducting an appropriateassay or measurement, including, e.g., quality of life assessments orsuitable assays for the level or activity of molecules, cells or cellmigration within a subject. Modulation may be transient, prolonged orpermanent or it may be variable at relevant times during or after aformula 1 compound is administered to a subject or is used in an assayor other method described herein or a cited reference, e.g., withinabout 1 hour of the administration or use of a formula 1 compound toabout 3, 6, 9 months or more after a subject(s) has received a formula 1compound.

Terms such as “antigen”, “immunogen” or the like mean a molecule thatcomprises one or more epitopes that are capable of stimulating asubject's immune system to make, e.g., a secretory, humoral or cellularantigen-specific response against the antigen or immunogen. These termsalso include fragments or synthetic or natural derivatives of thesemolecules that retain at least a detectable capacity, e.g., at leastabout 10%, 20%, 30%, 40%, 50% or more of the native antigen's antigeniccapacity, to stimulate a subject's immune system in a desired manner.

“Vaccine composition”, “vaccine” or similar terms mean an agent suitablefor stimulating a subject's immune system to ameliorate a currentcondition or to protect against or to reduce present or future harm orinfection, e.g., reduced tumor cell proliferation or survival, reducedpathogen replication or spread in a subject or a detectably reducedunwanted symptom(s) associated with a condition. Vaccines may modulate,typically detectably enhance, humoral, cell mediated or innate immuneresponses.

“Immunization” means the process of inducing a detectable and continuingmoderate or high level of antibody or cellular immune response that isdirected against an antigen to which the subject has been exposed. Suchresponses are typically detectably maintained for at least about 3-48months or more.

At various locations in the present disclosure, e.g., in any disclosedembodiments or in the claims, reference is made to compounds,compositions, formulations, or methods that comprise one or morespecified components, elements or steps. Invention embodiments alsospecifically include those compounds, compositions, formulations ormethods that consist of or that consist essentially of those specifiedcomponents, elements or steps. The terms “comprising”, “consist of” and“consist essentially of” have their normally accepted meanings underU.S. patent law. For example, disclosed compositions or methods that“comprise” a component or step are open and they include or read onthose compositions or methods plus an additional component(s) orstep(s). Similarly, disclosed compositions or methods that “consist of”a component or step are closed and they would not include or read onthose compositions or methods having appreciable amounts of anadditional component(s) or an additional step(s).

“Alkyl” as used here means linked normal, secondary, tertiary or cycliccarbon atoms, i.e., linear, branched, cyclic or any combination thereof.Alkyl moieties, as used herein, may be saturated, or unsaturated, i.e.,the moiety may comprise one or more independently selected double bondsor triple bonds. Unsaturated alkyl moieties include moieties asdescribed for alkenyl and alkynyl moieties described below. The numberof carbon atoms in an alkyl group or moiety is 1 to about 50, e.g.,about 1-30 or about 1-20, unless otherwise specified, e.g., C₁₋₈ alkylmeans an alkyl moiety containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms.When an alkyl group is specified, species may include methyl, ethyl,1-propyl (n-propyl), 2-propyl (i-propyl, —CH(CH₃)₂), 1-butyl (n-butyl),2-methyl-1-propyl (i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl (—CH(CH₂CH₃)₂)_(,)2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl (—CH(CH₃)CH(CH₃)₂),3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl, 2-hexyl (—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl(—CH(CH₂CH₃)(CH₂CH₂CH₃)), 2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃),3-methyl-2-pentyl (—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl(—CH(CH₃)CH₂CH(CH₃)₂), 3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂),2-methyl-3-pentyl (—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl(—C(CH₃)₂CH(CH₃)₂), 3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃), cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,—(CH₂)_(n)—(CHCH₃)_(m)—(CH₂)_(o)—CH₃ and—(CH₂)_(n)—(CHC₂H₅)_(m)—(CH₂)_(o)—CH₃ where n, m and o independently are0, 1, 2, 3, 4, 5, 6, 7 or 8.

“Alkenyl” as used here means a moiety that comprises linked normal,secondary, tertiary or cyclic carbon atoms, i.e., linear, branched,cyclic or any combination thereof, that comprises one or more doublebonds (e.g., —CH═CH—), e.g., 1, 2, 3, 4, 5, 6 or more, typically 1 or 2.The number of carbon atoms in an alkenyl group or moiety is 2 to about50, e.g., about 2-30 or about 2-20, unless otherwise specified, e.g.,C₂₋₈ alkenyl or C2-8 alkenyl means an alkenyl moiety containing 2, 3, 4,5, 6, 7 or 8 carbon atoms. When an alkenyl group is specified, speciesmay include vinyl, allyl, —(CH₂)_(n)—(CH═CH)—(CH₂)_(m)—CH₃,—(CH₂)_(n)—(CCH₃═CH)—(CH₂)_(m)—CH₃, —(CH₂)_(n)—(CH═CCH₃)—(CH₂)_(m)—CH₃and —(CH₂)_(n)—(CH═CH)₀₋₁—(CH₂)_(m)—CH₂CH═CH₂, where n and mindependently are 0, 1, 2, 3, 4, 5, 6, 7 or 8.

“Alkynyl” as used here means a moiety that comprises linked normal,secondary, tertiary or cyclic carbon atoms, i.e., linear, branched,cyclic or any combination thereof, that comprises one or more triplebonds (—C═C—), e.g., 1, 2, 3, 4, 5, 6 or more, typically 1 or 2 triplebonds, optionally comprising 1, 2, 3, 4, 5, 6 or more double bonds, withthe remaining bonds being single bonds. The number of carbon atoms in analkenyl group or moiety is 2 to about 50, e.g., about 2-30 or about2-20, unless otherwise specified, e.g., C₂₋₈ alkynyl or C₂₋₈ alkynylmeans an alkynyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms.When an alkynyl group is specified, groups and species may include —C≡H,—CCCH₃, —CCCH₂CH₃, —CCC₃H₇, —CCCH₂C₃H₇, —(CH₂)_(n)—(C≡C)—(CH₂)_(m)—CH₃,and —(CH₂)_(n)—(C≡C)₀₋₁—(CH₂)_(m)—CH₂C≡CH, where n and m independentlyare 0, 1, 2, 3, 4, 5, 6, 7 or 8.

“Aryl” means phenyl or naphthyl.

“Substituted alkyl”, “substituted alkenyl”, “substituted heterocycle”,“substituted aryl”, “substituted monosaccharide” and the like mean analkyl, alkenyl, heterocycle, aryl, monosaccharide or other group ormoiety as defined herein that has a substituent(s) or that comprises asubstituent(s) that replaces a hydrogen atom(s) and is bonded to acarbon atom(s) or a substituent(s) that interrupts a carbon atom chain.Substituted heterocycles may have a substituent bonded to a ring carbonor a ring heteroatom such as a nitrogen. Substituents include 1, 2, 3,4, 5, 6 or more independently selected —O—, —S—, —NH—, —C(O)—,—C(O)OR¹⁵, —C(O)OR^(PR), —C(O)SR¹⁵, —C(O)SR^(PR), —CHO, —CH₂SH, —C═N—,—OH, —OR¹⁵, —OR^(PR), —C(O)OR^(PR), —C(O)CH₃, —C(S)CH₃, —C(S)SH,—C(S)SR¹⁵, —C(S)SR^(PR), —C(O)CH₂OH, —C(O)CH₂F, —C(O)CH₂Cl, —C(O)CH₂Br,—C(O)CH₂I, —C(O)NHCH₃, —C(O)NHC₂H₅, —C(O)NHC(CH₃)₃, —O—CH₂—C(O)—C(CH₃)₃,—C(O)—C(CH₃)₃, —O—CH(CH₃)—O—C(CH₃)₃, —C(O)O—, —C(S)OR^(PR), —C(S)O—,—OC(O)—, —C(O)H, —OCH₂—, —CH₂—O—CH₂—, —(CH₂)₁₋₂—O(CH₂)₂, —OCH₂CH₂—,—OCH₂O—, —OCH₂CH₂O—, —CH₂OH, —CH₂F, —CHF₂, —CF₃, —CH₂Cl, —CH₂Br, —CH₂I,—C₂H₄Cl, —C₂H₄Br, —C₂H₄I, —CH₂CH₂F, —CH₂CHF₂, —CH₂CF₃, —N(R^(PR))₂,—NHR^(PR), —NHC(O)—, —CH₂—NR^(PR)—, —CH₂—NHR^(PR), —CH₂—NHC(O)—,—C(O)NH—, —C(O)NHR^(PR), —OC(O)NR^(PR)—, —OC(O)NHR^(PR), —C(═NH)—NH₂,—C(═NH)OH, —C(═N—NH₂)OH, —C(O)NHOH, —NH₂, —NHR^(PR), ═NOH, —NHR¹⁵,═NR¹⁵, ═N—, —NR^(PR)C(O)NR^(PR)—, —NR^(PR)C(O)NHR^(PR), —NR^(PR)CH₂—,—NR^(PR)CH₂CH₂—, —NO₂, —S—, —SR¹⁵, —SR^(PR), —S(O)R¹⁵, —S(O)OR¹⁵,—S(O)—, —S(O)(O)—, —S(S)(O)—, —S(O)(O)—O—, —S(S)(O)—O—, —S(S)(S)—O—,—S(O)OR^(PR), —S(O)(O)OH, —S(O)(O)OR¹⁵, —S(O)(O)OR^(PR), —S(O)OH,—S(O)OR¹⁵, —S(O)OR^(PR), —S(O)R¹⁵, —S(O)R^(PR), —S(S)OH, —S(O)SH,—S(O)SR¹⁵, —CN, —SCN, —NO₂, —C(O)OH, —C(O)OR¹⁵, —C(O)OR^(PR), —C(O)SH,—C(O)SR¹⁵, —C(O)SR^(PR), —C(S)OH, —C(S)OR¹⁵, —C(S)OR^(PR), —O—P(O)(O)OH,—O—P(O)(O)OR¹⁵, —O—P(O)(O)OR^(PR), —O—P(S)(O)OH, —O—P(S)(O)OR¹⁵,—O—P(S)(O)OR^(PR), —O—P(O)(O)SH, —O—P(O)(O)SR¹⁵, —O—P(O)(O)SR^(PR), —F,—Cl, —Br, —I, -amino acid-, —O-monosaccharide, —O-disaccharide,—S-monosaccharide, —S-disaccharide, a polymer, e.g., a PEG, andcombinations of these moieties and salts on any of these moieties thatcan form a salt, where R^(PR) independently is hydrogen, a protectinggroup or both R^(PR) are hydrogen or together are a protecting group andR¹⁵ is —H, —CH₃, —C₂H₅, —C₃H₇, —C₄H₉, —C(CH₃)₃, —CH₂OH, —C₂H₄OH,—C₃H₆OH, —C₄H₈OH—C(CH₂OH)(CH₃)₂, —C₃H₅, —C₄H₇, optionally substitutedC1-10 alkyl, C1-10 perfluoroalkyl, optionally substituted aryl,optionally substituted C1-12 alkylaryl, optionally substituted C1-12arylalkyl, optionally substituted allyl, optionally substitutedheterocycle, optionally substituted C1-4 alkyl-optionally substitutedheterocycle or optionally substituted heterocycle-optionally substitutedC1-4 alkyl. Substituents are independently chosen when more than one ispresent. Alkenyl and alkynyl groups that comprise a substituent(s), areoptionally substituted at a carbon that is one or more methylene moietyremoved from the double bond, e.g., separated by one, two, three or moreindependently selected —CH₂—, —CH(C₁₋₆ optionally substituted alkyl)-,—CH(C₁₋₆ optionally substituted alkenyl)-, —CH(C₁₋₆ optionallysubstituted alkynyl)-, —CH(optionally substituted heterocycle)-,—CH(optionally substituted aryl-optionally substituted alkyl)- or—CH(optionally substituted alkyl-optionally substituted aryl)- moieties.

“Heterocycle” or “heterocyclic” includes by way of example and notlimitation the heterocycles described in Paquette, Leo A.; “Principlesof Modern Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968),particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. 1960, 82:5566. Heterocycles are typically bondedto moieties of which they are a part through a ring carbon atom, a ringnitrogen atom or a ring sulfur atom.

Examples of heterocycles include by way of example and not limitationpyridyl, thiazolyl, tetrahydrothiophenyl, sulfur oxidizedtetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl,indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl,piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl,6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2dithiazinyl, thienyl, thianthrenyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl,2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Typically, nitrogen bonded heterocyclesinclude 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl,and 1-piperidinyl.

“Heteroaryl” means an aromatic ring or two or more fused rings thatcontain one or more aromatic rings where the ring or fused ringscomprise 1, 2, 3 or more heteroatoms, usually oxygen (—O—), nitrogen(—NX—) or sulfur (—S—) where X is —H, a protecting group or C₁₋₆ alkyl,usually —H. Examples are as described for heterocycle.

“Alcohol” as used herein means an alcohol that comprises a C₁₋₁₂ alkylmoiety substituted at a hydrogen atom with one hydroxyl group. Alcoholsinclude methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol,s-butanol, t-butanol, n-pentanol, i-pentanol, n-hexanol, cyclohexanol,n-heptanol, n-octanol, n-nonanol and n-decanol. The carbon atoms inalcohols can be straight, branched or cyclic. Alcohol includes anysubset of the foregoing, e.g., C₂₋₄ alcohols (alcohols having 2, 3 or 4carbon atoms).

“Halogen” means fluorine, chlorine, bromine or iodine.

“Protecting group” means a moiety that prevents the atom to which it islinked from participating in unwanted reactions. For example, for—OR^(PR), R^(PR) may be hydrogen or a protecting group for the oxygenatom found in a hydroxyl, while for —C(O)—OR^(PR), R^(PR) may behydrogen or a carboxyl protecting group, for —SR^(PR), R^(PR) may behydrogen or a protecting group for sulfur in thiols for instance, andfor —NHR^(PR) or —N(R^(PR))₂—, R^(PR) may be hydrogen or a nitrogen atomprotecting group for primary or secondary amines. Hydroxyl, amine andother reactive groups are found in formula 1 compounds at, e.g., R¹ orR². These groups may require protection against reactions taking placeelsewhere in the molecule. The protecting groups for oxygen, sulfur ornitrogen atoms are usually used to prevent unwanted reactions withelectrophilic compounds, such as acylating used, e.g., in steroidchemistry.

“Ester” means a moiety that comprises a —C(O)—O— structure. Typically,esters as used here comprise an organic moiety containing about 1-50carbon atoms (e.g., about 2-20 carbon atoms) and 0 to about 10independently selected heteroatoms (e.g., O, S, N, P, Si), where theorganic moiety is bonded to a formula 1 steroid nucleus at, e.g., R¹ orR² through the —C(O)—O— structure, e.g., organic moiety-C(O)—O-steroidor organic moiety-O—C(O)-steroid. The organic moiety usually comprisesone or more of any of the organic groups described above, e.g., C₁₋₂₀alkyl moieties, C₂₋₂₀ alkenyl moieties, C₂₋₂₀ alkynyl moieties, arylmoieties, C₂₋₉ heterocycles or substituted derivatives of any of these,e.g., comprising 1, 2, 3, 4 or more substituents, where each substituentis independently chosen. Exemplary substitutions for hydrogen or carbonatoms in these organic groups are as described above for substitutedalkyl moieties and include 1, 2, 3, 4, 5, 6 or more, usually 1, 2, or 3—O—, —S—, —NR^(PR)— (including —NH—), —C(O)—, —CHO, —CHS, —C═NH, —C(S),═O, ═S, —N(R^(PR))₂ (including —NH₂), —C(O)OR^(PR) (including —C(O)OH),—OC(O)R^(PR) (including —O—C(O)—H), —OR^(PR) (including —OH), —SR^(PR)(including —SH), —NO₂, —CN, —SCN, —C₆H₅, —CH₂C₆H₅, —NHC(O)—, —C(O)NH—,—OC(O)—, —C(O)O—, —O-A8, —S-A8, —C(O)-A8, —OC(O)-A8, —C(O)O-A8, ═N—,—N═, ═N—OH, —OPO₃(R^(PR))₂, —OSO₃H₂ or halogen moieties or atoms, whereeach R^(PR) is —H, an independently selected protecting group or bothR^(PR) together comprise a protecting group, and A8 is C₁₋₈ alkyl, C₂₋₈alkenyl, C₂₋₈ alkynyl, C₁₋₄ alkyl-aryl (e.g., benzyl), aryl (e.g.phenyl) or C₀₋₄ alkyl-C₂₋₉ heterocycle. Substitutions are independentlychosen. The organic moiety includes compounds defined by the R₄variable. The organic moieties exclude obviously unstable moieties,e.g., —O—O—, except where such unstable moieties are transient speciesthat one can use to make a compound with sufficient chemical stabilityfor one or more of the uses described herein, including for synthesis ofthe formula 1 or other compounds. The substitutions listed above aretypically substituents that one can use to replace one or more carbonatoms, e.g., —O— or —C(O)—, or one or more hydrogen atom, e.g., halogen,—NH₂ or —OH. Exemplary esters include one or more independently selectedacetate, enanthate, propionate, isopropionate, cyclopropionate,isobutyrate, butyrate, valerate, caproate, isocaproate, hexanoate,heptanoate, octanoate, nonanoate, decanoate, undecanoate, phenylacetateor benzoate, which are typically hydroxyl esters.

“Thioester” means a moiety that comprises a —C(O)—S— structure.Typically, thioesters as used here comprise an organic moiety containingabout 1-50 carbon atoms (e.g., about 2-20 carbon atoms) and 0 to about10 heteroatoms (e.g., O, S, N, P, Si), where the organic moiety isbonded to a formula 1 steroid nucleus at a variable group such as R¹,R², R³, R⁴ or R¹⁰ through the —C(S)—O— structure, e.g., organicmoiety-C(S)—O-steroid or organic moiety-O—C(S)-steroid. The organicmoiety is as described above for esters.

“Thionoester” means a moiety that comprises a —C(S)—O— structure.Typically, thionoesters as used here comprise an organic moietycontaining about 1-50 carbon atoms (e.g., about 2-20 carbon atoms) and 0to about 10 heteroatoms (e.g., O, S, N, P, Si), where the organic moietyis bonded to a formula 1 steroid nucleus at a variable group such as R¹,R², R³, R⁴ or R¹⁰ through the —C(S)—O— structure, e.g., organicmoiety-C(S)—O-steroid or organic moiety-O—C(S)-steroid. The organicmoiety is as described above for esters.

“Acetal” means a moiety that comprises (1) a —O—[C(CR³⁶)₂]₁₋₄—O—structure where the open valences are bonded to adjacent carbons on thesteroid nucleus, e.g., the 16 and 17 positions or the 2 and 3 positions,or (2) a —O—[C(CR³⁶)₂]₁₋₄—O— structure where the open valences arebonded to the same carbon on the steroid nucleus, where each R³⁶independently is —H, —F, —Cl, —Br, —I or an organic moiety such as C1-6alkyl (e.g., methyl or ethyl), C2-6 alkenyl, aryl or a heterocycle, anyof which are optionally substituted, e.g., —CF₃ or —CH₂OH. Typically,acetals as used here comprise an organic moiety containing about 1-50carbon atoms (e.g., about 2-20 carbon atoms) and 0 to about 10heteroatoms (e.g., O, S, N, P, Si), where the organic moiety is bondedto a formula 1 steroid nucleus at variable groups such as R¹, R², R³, R⁴or R¹⁰ through the —O—[C(CR³⁶)₂]₁₋₄—O— structure, e.g.,16-steroid-O—[C(CR³⁶)₂]₁₋₄—O-17-steroid or17-steroid-O—[C(CR³⁶)₂]₁₋₄—O-17-steroid. The organic moiety is asdescribed above for esters.

“Ketal” and “thioketal” mean an organic moiety that is bonded to twoadjacent steroid ring atoms in the formula 1 compounds, e.g., ring atomsat the 1-2, 2-3, 3-4, 6-7, 14-15, 15-16 or 16-17 positions. The steroidring atoms are carbon and the ketal is bonded to each adjacent carbon byan oxygen atom. Thioketals are bonded through one oxygen and one sulfuratom. One, two or more of two adjacent R¹-R⁶ and R¹⁰ may comprise anindependently selected ketal or thioketal in any of the formula 1compounds disclosed herein. The oxygen or sulfur atoms in ketals andthioketals are linked by an optionally substituted alkyl moiety.Typically the alkyl moiety is an optionally substituted C1-C6 alkylenesuch as —C(CH₃)₂—, —CH(CH₃)—, —CH₂—, —CH₂—CH₂—, —C(C₂-C₄ alkyl)₂- or—CH(C₂-C₄ alkyl)-. Exemplary ketal and thioketals include —O—C(CH₃)₂—O—,—O—C(CH₃)(heterocycle)-O—, —O—CH(heterocycle)-O—, —O—C(CH₃)(aryl)-O—,—O—CH(aryl)-O—, —S—C(CH₃)₂—O—, —O—CH₂—CH₂—O—, —O—C(CH₃)₂—CH₂—O—,—O—C(CH₃)₂—C(CH₃)₂—O—, —S—C(CH₃)₂—CH₂—O—, —O—C(CH₃)₂—CH₂—S— and thelike.

“Thioacetal” means a moiety that comprises (1) a —S—[C(CR³⁶)₂]₁₋₄—O— or—S—[C(CR³⁶)₂]₁₋₄—S— structure where the open valences are bonded toadjacent carbons on the steroid nucleus, e.g., the 16 and 17 positionsor the 2 and 3 positions, or (2) a —S—[C(CR³⁶)₂]₁₋₄—O— or—S—[C(CR³⁶)₂]₁₋₄—S— structure where the open valences are bonded to thesame carbon on the steroid nucleus, where each R³⁶ independently is —H,—F, —Cl, —Br, —I or an organic moiety such as C1-6 alkyl (e.g., methylor ethyl), C2-6 alkenyl, aryl or a heterocycle, any of which areoptionally substituted, e.g., —CF₃ or —CH₂OH. Typically, thioacetals asused here comprise an organic moiety containing about 1-50 carbon atoms(e.g., about 2-20 carbon atoms) and 0 to about 10 heteroatoms (e.g., O,S, N, P, Si), where the organic moiety is bonded to a formula 1 steroidnucleus at variable groups such as R¹, R², R³, R⁴ or R¹⁰ through the—S—[C(CR³⁶)₂]₁₋₄—O— or —S—[C(CR³⁶)₂]₁₋₄—S— structure, e.g.,16-steroid-S—[C(CR³⁶)₂]₁₋₄—O-17-steroid,16-steroid-O—[C(CR³⁶)₂]₁₋₄—S-17-steroid,16-steroid-S—[C(CR³⁶)₂]₁₋₄—S-17-steroid,17-steroid-S—[C(CR³⁶)₂]₁₋₄—O-17-steroid, organic moiety-S—C(O)-steroidor steroid-S—C(O)-organic moiety. The organic moiety is as describedabove for esters.

“Phosphoester” or “phosphate ester” means a moiety that comprises a—O—P(OR^(PR))(O)—O— structure where R^(PR) is hydrogen (—H), aprotecting group or an organic moiety as described for esters.Typically, phosphoesters as used here comprise a hydrogen atom, aprotecting group or an organic moiety containing about 1-50 carbon atomsand 0 to about 10 heteroatoms (e.g., O, S, N, P, Si) linked to a formula1 steroid nucleus at a variable group such as R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ orR¹⁸ through the —O—P(O)(O)—O— structure, e.g., organicmoiety-O—P(O)(OH)—O-steroid. The organic moiety is as described abovefor esters.

“Phosphothioester” means a moiety that comprises a—O—P(SR^(PR))(O)-structure where R^(PR) is —H, a protecting group or anorganic moiety as described for esters. Typically, phosphothioesters asused here comprise a hydrogen atom, a protecting group or an organicmoiety containing about 1-50 carbon atoms and 0 to about 10 heteroatoms(e.g., O, S, N, P, Si) linked to a formula 1 steroid nucleus at avariable group such as R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸ through the—O—P(O)(O)—O— structure, e.g., organic moiety-O—P(O)(SH)—O-steroid. Theorganic moiety is as described above for esters.

“Phosphonoester” means a moiety that comprises a —P(OR^(PR))(O)—structure where R^(PR) is —H, a protecting group or an organic moiety asdescribed for esters. Typically, phosphonoesters as used here comprise ahydrogen atom, a protecting group or an organic moiety containing about1-50 carbon atoms and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si)linked to a formula 1 steroid nucleus at a variable group such as R¹-R⁶,R¹⁰, R¹⁵, R¹⁷ or R¹⁸ through the —P(OR^(PR))(O)—O— structure, i.e.,organic moiety-P(OR^(PR))(O)—O-steroid orsteroid-P(OR^(PR))(O)—O-organic moiety. The organic moiety is asdescribed above for esters.

“Phosphiniester” means a moiety that comprises a —P(O)H— structure whereR^(PR) is —H, a protecting group or an organic moiety as described foresters. Typically, phosphiniesters as used here comprise a hydrogenatom, a protecting group or an organic moiety containing about 1-50carbon atoms and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si) linkedto a formula 1 steroid nucleus at a variable group such as R¹-R⁶, R¹⁰,R¹⁵, R¹⁷ or R¹⁸ through the —P(O)H— structure, i.e., organicmoiety-P(O)H-steroid or steroid-P(O)H-organic moiety. The organic moietyis as described above for esters.

“Sulfate ester” means a moiety that comprises a —O—S(O)(O)—O— structure.Typically, sulfate esters as used here comprise a hydrogen atom, aprotecting group or an organic moiety containing about 1-50 carbon atomsand 0 to about 10 heteroatoms (e.g., O, S, N, P, Si) linked to a formula1 steroid nucleus at a variable group such as R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ orR¹⁸ through the —O—S(O)(O)—O— structure, e.g., organicmoiety-O—S(O)(O)—O-steroid. The organic moiety is as described above foresters.

“Sulfite ester” means a moiety that comprises a —O—S(O)—O— structure.Typically, sulfite esters as used here comprise an organic moietycontaining about 1-50 carbon atoms and 0 to about 10 heteroatoms (e.g.,O, S, N, P, Si) linked to a formula 1 steroid nucleus at a variablegroup such as R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸ through the—O—S(O)—O-structure, e.g., organic moiety-O—S(O)—O-steroid. The organicmoiety is as described above for esters.

“Amide” means an organic moiety as described for ester that comprises 1,2, 3, 4 or more —C(O)—NR^(PR)— moieties, usually 1 or 2, where R^(PR) is—H or a protecting group, R^(PR) is usually H. In some embodiments, the—C(O)NR^(PR)— group is linked to the steroid nucleus at a variable groupsuch as R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸, i.e., organicmoiety-C(O)NR^(PR)-steroid or steroid-C(O)NR^(PR)-organic moiety. Theorganic moiety is as described above for esters.

“Ether” means an organic moiety as described for ester that comprises 1,2, 3, 4 or more —O— moieties, usually 1 or 2. In some embodiments, the—O— group is linked to the steroid nucleus at a variable group such asR¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸, e.g., organic moiety-O-steroid. The organicmoiety is as described above for esters.

“Thioether” means an organic moiety as described for ester thatcomprises 1, 2, 3, 4 or more —S— moieties, usually 1 or 2. In someembodiments, the —S— group is linked to the steroid nucleus at avariable group such as R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸, e.g., organicmoiety-S-steroid. The organic moiety is as described above for esters.

“Acyl group” means an organic moiety as described for ester thatcomprises 1, 2, 3, 4 or more —C(O)— groups. In some embodiments, the—C(O)— group is linked to the steroid nucleus at a variable group suchas R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸, e.g., organic moiety-C(O)-steroid. Theorganic moiety is as described above for esters.

“Thioacyl” means an organic moiety as described for ester that comprises1, 2, 3, 4 or more —C(S)— groups. In some embodiments, the —C(S)— groupis linked to the steroid nucleus at a variable group such as R¹-R⁶, R¹⁰,R¹⁵, R¹⁷ or R¹⁸, e.g., organic moiety-C(S)-steroid. The organic moietyis as described above for esters.

“Carbonate” means an organic moiety as described for ester thatcomprises 1, 2, 3, 4 or more —O—C(O)—O— structures. Typically, carbonategroups as used here comprise an organic moiety containing about 1-50carbon atoms and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si) linkedto a formula 1 steroid nucleus at a variable group such as R¹-R⁶, R¹⁰,R¹⁵, R¹⁷ or R¹⁸ through the —O—C(O)—O— structure, e.g., organicmoiety-O—C(O)—O-steroid. The organic moiety is as described above foresters.

“Carbamate” means an organic moiety as described for ester thatcomprises 1, 2, 3, 4 or more —O—C(O)NR^(PR)— structures where R^(PR) is—H, a protecting group or an organic moiety as described for ester.Typically, carbamate groups as used here comprise an organic moietycontaining about 1-50 carbon atoms and 0 to about 10 heteroatoms (e.g.,O, S, N, P, Si) linked to a formula 1 steroid nucleus at a variablegroup such as R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸ through the —O—C(O)—NR^(PR)—structure, e.g., organic moiety-O—C(O)—NR^(PR)-steroid orsteroid-O—C(O)—NR^(PR)-organic moiety. The organic moiety is asdescribed above for esters.

As used herein, “monosaccharide” means a polyhydroxy aldehyde or ketonehaving the empirical formula (CH₂O)_(n) where n is 3, 4, 5, 6 or 7.Monosaccharide includes open chain and closed chain forms, but willusually be closed chain forms. Monosaccharide includes hexofuranose andpentofuranose sugars such as 2′-deoxyribose, ribose, arabinose, xylose,their 2′-deoxy and 3′-deoxy derivatives and their 2′,3′-dideoxyderivatives. Monosaccharide also includes the 2′,3′ dideoxydidehydroderivative of ribose. Monosaccharides include the D-, L- and DL-isomersof glucose, fructose, mannose, idose, galactose, allose, gulose,altrose, talose, fucose, erythrose, threose, lyxose, erythrulose,ribulose, xylulose, ribose, arabinose, xylose, psicose, sorbose,tagatose, glyceraldehyde, dihydroxyacetone and their monodeoxy or otherderivatives such as rhamnose and glucuronic acid or a salt of glucuronicacid. Monosaccharides are optionally protected or partially protected.Exemplary monosacharides include

where R³⁷ independently is hydrogen, a protecting group, acetamido(—NH—Ac), optionally substituted alkyl such as methyl or ethyl, or anester such as acetate or proprionate, R³⁸ is hydrogen, hydroxyl, —NH₂,—NHR^(PR), optionally substituted alkyl such as methyl or ethyl, or acation such as NH₄ ⁺, Na⁺ or K⁺ and R³⁹ is hydrogen, hydroxyl, acetate,proprionate, optionally substituted alkyl such as methyl, ethyl, methoxyor ethoxy.

Optionally substituted alkyl group, optionally substituted alkenylgroup, optionally substituted alkynyl group, optionally substituted arylmoiety and optionally substituted heterocycle mean an alkyl, alkenyl,alkynyl, aryl or heterocycle moiety that contains an optionalsubstitution(s). Such moieties include C₁₋₂₀ alkyl moieties, C₂₋₂₀alkenyl moieties, C₂₋₂₀ alkynyl moieties, aryl moieties, C₂₋₉heterocycles or substituted derivatives of any of these. Typicalsubstitutions for these organic groups are as described above forsubstituted alkyl moieties and include, e.g., 1, 2, 3, 4, 5, 6 or more,independently selected —O—, —S—, —NR^(PR)—, —C(O)—, —N(R^(PR))₂,—C(O)OR^(PR), —OC(O)R^(PR), —OR^(PR), —SR^(PR), —NO₂, —CN, —NHC(O)—,—C(O)NH—, —OC(O)—, —C(O)O—, —O-A8, —S-A8, —C(O)-A8, —OC(O)-A8,—C(O)O-A8, ═N—, —N═, —OPO₂R^(PR), —OSO₃H or halogen moieties or atoms,where R^(PR) independently is —H, a protecting group or both R^(PR)together are a protecting group and A8 is C₁₋₈ alkyl, C₁₋₈ alkenyl, C₁₋₈alkynyl, C₁₋₄ alkyl-aryl (e.g., benzyl), aryl (e.g. phenyl) or C₁₋₄alkyl-C₁₋₅ heterocycle. Substitutions are independently chosen. Theorganic moieties as described here, and for other any other moietiesdescribed herein, exclude obviously unstable moieties, e.g., —O—O—,except where such unstable moieties are transient species that one canuse to make a compound with sufficient chemical stability for the one ormore of the uses described herein.

Optionally substituted “monosaccharide” comprise any C3-C7 sugar, D-, L-or DL-configurations, e.g., erythrose, glycerol, ribose, deoxyribose,arabinose, glucose, mannose, galactose, fucose, mannose, glucosamine,N-acetylneuraminic acid, N-acetylglucosamine, N-acetylgalactosamine thatis optionally substituted at one or more hydroxyl groups. Suitablesubstitutions are as described above for substituted alkyl moieties andinclude independently selected hydrogen, hydroxyl, protected hydroxyl,carboxyl, azido, cyano, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —O—C₂₋₆ alkenyl,—S—C₂₋₆ alkenyl, optionally protected amine, optionally protectedcarboxyl, halogen, thiol or protected thiol. The linkage between themonosaccharide and the steroid is α or β.

Optionally substituted “oligosaccharide” comprises two, three, four ormore of any C3-C7 sugars that are covalently linked to each other. Thelinked sugars may have D-, L- or DL-configurations. Suitable sugars andsubstitutions are as described for monosaccharides. The linkage betweenthe oligosaccharide and the steroid is α or β, as are the linkagesbetween the monosaccharides that comprise the oligosaccharide.

Nucleoside includes 3TC, AZT, D4T, ddI, ddC, G, A, U, C, T, dG, dA, dTand dC.

Polymer includes biocompatible organic polymers, e.g., PEGs andpolyhydroxyalkyl polymers.

PEG means an ethylene glycol polymer that contains about 20 to about2000000 linked monomers, typically about 50-1000 linked monomers,usually about 100-300. Polyethylene glycols include PEGs containingvarious numbers of linked monomers or having differing average molecularweights, e.g., PEG20, PEG30, PEG40, PEG60, PEG80, PEG100, PEG115,PEG200, PEG300, PEG400, PEG500, PEG600, PEG1000, PEG1500, PEG2000, PEG3350, PEG4000, PEG4600, PEG5000, PEG6000, PEG8000, PEG11000, PEG12000,PEG2000000 and any mixtures thereof.

As used herein, position numbers that are given for the formula 1compounds use the numbering convention for cholesterol.

“Spiro ring” or “spiro structure” and similar terms mean cyclicstructures that comprise 4, 5, 6, 7 or 8 ring members, i.e., they are4-, 5-, 6-, 7- or 8-sided. In some embodiments, spiro structures share acarbon atom that is present in the steroid ring system, e.g., at the 2,3, 7, 11, 15, 16 or 17 positions of the formula 1 compounds. Spirostructures include lactone rings or cyclic esters. Such spirolactonesinclude 5 and 6 membered rings, e.g., a spiro compound with a spiro ringat the 17 position such as

wherein X is —C(R¹⁰)₂— or —CHR¹⁰— and wherein independently selected R¹⁰groups are bonded to the 1-, 4-, 5-, 6-, 8-, 9-, 12-, and 14-positions.In some of these embodiments, the R¹⁰ variable group independently is—H, —OH, —OCH₃, —CH₃ or an optionally substituted alkyl.

Unless otherwise stated or implied by context, expressions of apercentage of a liquid ingredient, e.g., an excipient, in an inventioncomposition or formulation mean the ingredient's percent by volume(v/v). Thus, 20% propylene glycol means 20% v/v propylene glycol ispresent in an invention composition or formulation. The amount ofexcipient indicated in invention compositions is not affected by theform used, e.g., NF or USP grade solvent or excipient. Thus, aninvention composition that comprises about 30% polyethylene glycol 300NF can instead comprise a USP counterpart, provided that otherlimitations, such as the amount of water present, are not exceeded.

As used herein, “innate immunity” refers to one or more componentstypically associated with nonspecific immune defense mechanisms in asubject. These components include the alternate complement pathway,e.g., Factor B, Factor D and properdin; NK cells, phagocytes (monocytes,macrophages), neutrophils, eosinophils, dendritic cells, fibrocytes;anti-microbial chemicals, e.g., one or more of defensins; physicalbarriers—skin, mucosal epithelium; or certain interleukins, chemokines,cytokines, lung or alveolar macrophage respiratory burst activity or alung surfactant protein such as surfactant protein A or surfactantprotein D. Innate immunity plays a role in resistance to intracellularparasite infections, e.g., white blood cell infection, a liverinfection, and other infections, e.g., lymph node infections. Detectableenhancement of innate immunity mechanism by formula 1 compounds ormethod described herein can also enhance phagolysosome fusion ormovement, which some pathogens, e.g., intracellular bacteria such asmycobacteria, or Listeria inhibit.

Terms such as “immune disregulation”, “immune disregulation condition”,“unwanted immune response” and the like mean that a subject has or issubject to developing an immune response that is not desirable or issuboptimal for the subject's condition. Such disregulation or unwantedresponses can arise from various clinical conditions or diseases or as aresult of treatment of such conditions or diseases, e.g., inflammation,autoimmunity, organ or tissue transplant rejection (e.g., allograft,xenograft), infections, cancers, chemotherapy treatments, trauma,allergy conditions or in conditions where a subject mounts a Th1, Tc1,Th2 or Tc2 immune response that is considered to be pathogenic,ineffective, insufficient or suboptimal. Immune disregulation conditionsare as described herein or in the cited references.

Terms such as “cellular response”, “cellular activity”, biologicalresponse”, “biological activity” and the like mean a response oractivity that is detectably modulated in response to the presence of aformula 1 compound. Such responses or activities can be direct effectsor indirect effects on one or more cellular activities or on theexpression or level of one or more molecules that the affected cell(s)bind, sequester, synthesize or respond to. Such responses or activitiesinclude a detectable change in the synthesis or level of one or morecytokines, growth factors, transcription factors (including receptorsand their cofactors), enzymes, Th1- or Th2-associated antibody subtyperesponses or the like. Typically, the cytokines, growth factors,transcription factors, enzymes or antibodies that are modulated areinvolved in the amelioration of a pathological condition or in theestablishment, maintenance or progression of a pathological condition.

As used herein, references to CD molecules, specific immune cellsubsets, immune responses and the like, generally use nomenclature thatapplies to molecules, cells or the like that are found in humans.Analogs or counterparts of such molecules, cells or the like in otherspecies may have a differing nomenclature, but are included in thisinvention. A description of the nomenclature and function of various CDmolecules and immune cell subsets are as found in the scientificliterature. References to Th0, Th1 or Th2 cells and references to Th1 orTh2 immune responses in the context of human patients refers to thehuman counterparts of the murine Th0, Th1 or Th2 immune cells orresponses. For reviews see, e.g., A. K. Abbas et al., editors, Cellularand Molecular Immunology, W.B. Saunders Company, third edition, 1997,ISBN 0-7216-4024-9, pages 4-469, and I. Kimber and M. K. Selgrade,editors, T Lymphocyte Subpopulations in Immunotoxicology, John Wiley &Sons Ltd., 1998, ISBN 0-471-97194-4, pages 1-53.

“Immunosuppressive molecule” means molecules such as cyclosporin,cyclohexamide, mitomycin C, adriamycin, taxol and amphotericin B. Thesemolecules tend to have toxicities toward the immune system and aredirectly or indirectly immunosuppressive, e.g., they are toxic todividing cells, they inhibit proliferation of immune cell precursors orthey can downregulate an otherwise desired or improved immune responseor condition.

“Steroid receptor” means a gene product, typically a protein monomer ordimer that can bind to a ligand, e.g., a natural steroid, a steroidanalog, or another ligand such as a formula 1 compound or a metabolicprecursor thereof or a metabolite thereof, a lipid, e.g., aprostaglandin, or the like. Steroid receptors include orphan steroidreceptors. Orphan steroid receptors are proteins for which the naturalligand or biological function is at least partially unknown. As usedhere, steroid receptors include homodimers, e.g., SXR and (CARβ)₂, andheterodimers, e.g., PXR-CARβ or RXR-CARβ. Steroid receptors also includeisoforms, e.g., PXR.1 and PXR.2 for the PXR receptor, and homologs ofthe steroid receptors, e.g., the homolog of CARβ known as MB67. Isoformsare typically generated by different splicing pathways for a nuclear RNAfrom one gene, while homologs are typically a distinct copy of a steroidreceptor gene, where the gene copy encodes only relatively smalldifferences compared to the reference steroid receptor gene product.Such differences are most often found in areas other than thedimerization region and the steroid binding region of the steroidreceptor's structure. Typically isoforms and homologs bind the same orsimilar ligands as the reference gene product or steroid receptor.Steroid receptors may be of human or animal origin, e.g., obtained fromcells, tissues or cDNA expression libraries derived from cells ortissues of any primate, rodent (including murine), avian, ovine, bovine,equine, canine or feline species or any of the species or any specieswithin any group (e.g., Family or Genus) of species mentioned elsewhereherein or in any reference cited herein. Modulation of steroid receptorsby formula 1 compounds can arise from (1) their direct interaction witha steroid receptor or a cofactor thereof or (2) indirect effects such as(A) detectably increased or decreased synthesis or level of the steroidreceptor or (B) generation of a signal or stimulus that leads todetectable modulation of one or more biological activities of thereceptor, e.g., detectable inhibition of steroid receptor mediated genetranscription or detectable enhancement of steroid receptor mediatedgene transcription.

In the context of a combination of molecules that includes a steroidreceptor and a formula 1 compound, “invention complexes” or “complexes”include a complex that comprises a steroid receptor and a formula 1compound and optionally other molecules. These other molecules include(i) a DNA recognition sequence (“DNARS” hereafter), i.e., a sequencethat the steroid receptor specifically recognizes and binds to and (ii)a transcription factor that can bind to the steroid receptor-formula 1compound complex. As used herein, these complexes can arise in cells invitro or in vivo, or in cell-free systems. Complexes include, forexample, steroid receptor heterodimer-formula 1 compound combinations,steroid receptor homodimer-formula 1 compound combinations, steroidreceptor monomer-formula 1 compound combinations, steroid receptorheterodimer-formula 1 compound-DNA (or DNARS) combinations, steroidreceptor homodimer-formula 1 compound-DNA (or DNARS) combinations,steroid receptor heterodimer-formula 1 compound-transcription factorcombinations, steroid receptor homodimer-formula 1compound-transcription factor combinations, steroid receptorheterodimer-formula 1 compound-DNA (or DNARS)-transcription factorcombinations and steroid receptor homodimer-formula 1 compound-DNA (orDNARS)-transcription factor combinations.

An “agonist” or an “antagonist” is a compound or composition thatrespectively, either detectably increases or decreases the activity of areceptor, an enzyme or another biological molecule, which can lead toincreased or decreased transcription of a regulated gene or to anothermeasurable effect. The increase or decrease in a receptor's or enzyme'sactivity will be an increase or a decrease of at least about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or a range about between anytwo of these values, for one or more measurable activities. Receptors,their accessory factors and associated transcription factors canmodulate transcription of their target gene(s) by detectably enhancingtranscription or decreasing it. Biological activities of receptors mayalso include modulating biological responses such as signal transductionwithin a cell or ion flux, e.g., sodium, potassium or calcium, acrosscell or organelle membranes.

Terms such as “biologically active metabolite” and the like meanderivatives of the formula 1 compounds that retain a detectable level,e.g., at least about 10%, at least about 20%, at least about 30% or atleast about 50%, of at least one desired activity of the parentcompound, e.g., antiinflammatory activity or stimulation of a desiredimmune response. Determination of a desired activity is accomplishedessentially as described herein. Such metabolites can be generated inthe gastrointestinal tract, in blood or in one or more subject tissues.Such metabolites are detected using standard analytical methods, e.g.,GC-MS analysis of an optionally radiolabeled formula 1 compound and itsmetabolites, in blood, urine or other biological samples after it isadministered to a subject by one or more routes as disclosed herein.Terms such as “metabolic precursor” of formula 1 compounds and the likecan include compounds that generate a detectable level of the formula 1compound or a detectable level, e.g., at least about 10%, at least about20%, at least about 30% or at least about 50%, of at least one desiredactivity of the formula 1 compound. Determination of a desired activityis accomplished essentially as described herein. Conversion of metabolicprecursors can occur in the gastrointestinal tract, in blood or in oneor more subject tissues.

“Amino acid” means an amino acid moiety that comprises anynaturally-occurring or synthetic amino acid residue, i.e., any moietycomprising at least one carboxyl and at least one amino residue directlylinked by one, two three or more carbon atoms, typically one (α) carbonatom. The nature and identity of the intervening structure locatedbetween the carboxyl and amino groups can have a variety of structuresincluding those described herein. Typically, amino acids linked to thesteroid through the amine group have sufficient conformation and lengthto be capable of autocatalytic hydrolysis of the amino acid-steroid bondand release of the steroid. This can occur when the free carboxyl isgenerated in vivo by deesterification, deamidation or peptidolyticcleavage of the precursor containing a linkage between the amino acid'samine group and the steroid. Hydrolysis of the bond between an aminoacid's carboxyl or amino group and the steroid can also occur bychemical or enzymatic activity, e.g., esterase cleavage or non-enzymatichydrolysis.

In general, the amino acids corresponding to the residues employed inthe formula 1 compounds are naturally occurring and have no significantpharmacological activity per se. However, optimal pharmacokineticactivity, (substantially complete hydrolysis upon hydrolysis of thedistal amide or ester bond) may be achieved by using non-naturallyoccurring amino acid residues. The intervening structure may be assimple as methylene when the amino acid residue is glycyl, orsubstituted methylene for other a amino acids. The structure ordinarilycontains up to about 5 carbon or heteroatoms in the direct linkagebetween the amino acid's carboxyl carbon and the amine nitrogen. Thus,amino acids can comprise intervening ethylene, propylene, butylene, orpentylene groups or their substituted analogs, such as for example,oxyesters or ethers in which oxygen replaces carbon and, as appropriate,hydrogen. An example of such an intervening structure would be—CH—O—C(R²²)(R²³)—, where R²² and R²³ are independently selectedhydrogen or organic moieties as described above for esters. In someembodiments one of R²² and R²³ is hydrogen and the other is a C2-20organic moiety. Typically the organic moieties contain about 1-20 carbonatoms and 0, 1, 2, 3, 4 or 5 independently selected heteroatoms, whichare typically selected from oxygen, nitrogen, sulfur and phosphorus. Ingeneral, fewer intervening atoms are used when more rapid hydrolysis isdesired, although larger structures are suitable if, e.g., they possesssufficient flexibility or have conformations to allow positioning of thecarboxyl group in proximity to the amino acid-steroid bond.

Ordinarily, R²² is —H, methyl or hydroxymethyl, usually —H, and R²³ is aside chain or group of a naturally occurring amino acid. Amino acid sidechains include analogs where the side chain is a C₁₋₁₅ homolog of thecorresponding natural compound, e.g., methylene, ethylene, propylene,butylene or a substituted derivative thereof, e.g., an alkyl, ether oralkoxy (e.g., methoxy, ethoxy, propoxy) substituted derivative. Ingeneral, for carboxyl-containing side chains, if the C atom of the sidechain carboxyl is linked by 5 or less atoms to the N then the carboxyloptionally will be blocked, e.g. by esterification or amidation whereinthe ester or amide bonds are hydrolyzable in vivo. R²² also is takentogether with R³⁰ to form a proline residue (—CH₂—)₃. Thus, R²³ isgenerally a side group such as —H, —CH₃, —CH(CH₃)₂, —CH₂—CH(CH₃)₂,—CHCH₃—CH₂—CH₃, —CH₂—C₆H₅, —CH₂CH₂—S—CH₃, —CH₂OH, —CH(OH)—CH₃, —CH₂—SH,—CH₂—C₆H₄OH, —CH₂—CO—NH₂, —CH₂—CH₂—CO—NH₂, —CH₂—COOH, —CH₂—CH₂—COOH,—(CH₂)₄—NH₂ and —(CH₂)₃—NH—C(NH₂)—NH₂. R²³ also includes1-guanidinoprop-3-yl, benzyl, 4-hydroxybenzyl, imidazol-4-yl,indol-3-yl, methoxyphenyl and ethoxyphenyl. The optimal R³⁰ group isreadily selected using routine assays.

In general, the amino acid residue has the structure shown in theformulas below. Ordinarily, n is 1 or 2, R²² is —H and R²³ is a moietycontaining one or more of the following groups: amino, carboxyl, amide,carboxyl ester, hydroxyl, C₆-C₇ aryl, ether (—O—), thioether (—S—), n-,s- or t-alkyl (C₁-C₆), guanidinyl, imidazolyl, indolyl, sulfhydryl,sulfoxide, and phosphoryl. The R²² and R²³ substituents can have a widevariety of structures including those disclosed herein, e.g., esters,ethers or carbonates.

When the amino acid residues contain one or more chiral centers, any ofthe D, L, meso, threo or erythro (as appropriate) racemates or mixturesthereof, fall within the scope of this invention. In general, if it isdesired to rely on non-enzymatic means of hydrolysis, D isomers shouldbe used. On the other hand, L isomers may be more versatile since theycan be susceptible to both non-enzymatic as well as potential targetedenzymatic hydrolysis, and are more efficiently transported by amino acidor dipeptidyl transport systems in the gastrointestinal tract.

Examples of suitable amino acid residues include the following: Glycyl;aminopolycarboxylic acids, e.g., aspartic acid, β-hydroxyaspartic acid,glutamic acid, β-hydroxyglutamic acid, β-methylaspartic acid,β-methylglutamic acid, β,β-dimethylaspartic acid, γ-hydroxyglutamicacid, β,γ-dihydroxyglutamic acid, β-phenylglutamic acid,γ-methyleneglutamic acid, 3-aminoadipic acid, 2-aminopimelic acid,2-aminosuberic acid and 2-aminosebacic acid residues; amino acid amidessuch as glutaminyl and asparaginyl; polyamino- orpolybasic-monocarboxylic acids such as arginine, lysine, β-aminoalanine,γ-aminobutyrine, ornithine, citruline, homoarginine, homocitrulline,5-hydroxy-2,6-diaminohexanoic acid (commonly, hydroxylysine, includingallohydroxylysine) and diaminobutyric acid residues; other basic aminoacid residues such as histidinyl; diaminodicarboxylic acids such asα,α′-diaminosuccinic acid, α,α′-diaminoglutaric acid, α,α′-diaminoadipicacid, α,α′-diaminopimelic acid, α,α′-diamino-β-hydroxypimelic acid,α,α′-diaminosuberic acid, α,α′-diaminoazelaic acid, andα,α′-diaminosebacic acid residues; imino acids such as proline, 4- or3-hydroxy-2-pyrrolidinecarboxylic acid (commonly, hydroxyproline,including allohydroxyproline), γ-methylproline, pipecolic acid,5-hydroxypipecolic acid, —N([CH₂]_(n)COOR^(PR))₂, wherein n is 1, 2, 3,4, 5 or 6 and R^(PR) is —H or a protecting group, andazetidine-2-carboxylic acid residues; a mono- or di-alkyl (typicallyC₁-C₈ branched or normal) amino acid such as alanine, valine, leucine,allylglycine, butyrine, norvaline, norleucine, heptyline,α-methylserine, α-amino-α-methyl-γ-hydroxyvaleric acid,α-amino-α-methyl-δ-hydroxyvaleric acid,α-amino-α-methyl-ε-hydroxycaproic acid, isovaline, α-methylglutamicacid, α-aminoisobutyric acid, α-aminodiethylacetic acid,α-aminodiisopropylacetic acid, α-aminodi-n-propylacetic acid,α-aminodiisobutylacetic acid, α-aminodi-n-butylacetic acid,α-aminoethylisopropylacetic acid, α-amino-n-propylacetic acid,α-aminodiisoamyacetic acid, α-methylaspartic acid, α-methylglutamicacid, 1-aminocyclopropane-1-carboxylic acid; isoleucine, alloisoleucine,tert-leucine, β-methyltryptophan and α-amino-β-ethyl-β-phenylpropionicacid residues; β-phenylserinyl; aliphatic α-amino-β-hydroxy acids suchas serine, β-hydroxyleucine, β-hydroxynorleucine, β-hydroxynorvaline,and α-amino-β-hydroxystearic acid residues; α-Amino, α-, γ-, δ- orε-hydroxy acids such as homoserine, γ-hydroxynorvaline,δ-hydroxynorvaline and epsilon-hydroxynorleucine residues; canavinyl andcanalinyl; γ-hydroxyornithinyl; 2-Hexosaminic acids such asD-glucosaminic acid or D-galactosaminic acid residues; α-amino-β-thiolssuch as penicillamine, β-thiolnorvaline or β-thiolbutyrine residues;other sulfur containing amino acid residues including cysteine;homocystine; β-phenylmethionine; methionine; S-allyl-L-cysteinesulfoxide; 2-thiolhistidine; cystathionine; and thiol ethers of cysteineor homocysteine; phenylalanine, tryptophan and ring-substituted a aminoacids such as the phenyl- or cyclohexylamino acids α-aminophenylaceticacid, α-aminocyclohexylacetic acid and α-amino-β-cyclohexylpropionicacid; phenylalanine analogues and derivatives comprising aryl, loweralkyl, hydroxy, guanidino, oxyalkylether, nitro, sulfur orhalo-substituted phenyl (e.g., tyrosine, methyltyrosine and o-chloro-,p-chloro-, 3,4-dichloro, o-, m- or p-methyl-, 2,4,6-trimethyl-,2-ethoxy-5-nitro, 2-hydroxy-5-nitro and p-nitro-phenylalanine); furyl-,thienyl-, pyridyl-, pyrimidinyl-, purine or naphthylalanines; andtryptophan analogues and derivatives including kynurenine,3-hydroxykynurenine, 2-hydroxytryptophan and 4-carboxytryptophanresidues; α-amino substituted amino acid residues including sarcosine(N-methylglycine), N-benzylglycine, N-methylalanine, N-benzylalanine,N-methylphenylalanine, N-benzylphenylalanine, N-methylvaline andN-benzylvaline; and α-Hydroxy and substituted α-hydroxy amino acidresidues including serine, threonine, allothreonine, phosphoserine andphosphothreonine residues.

Any one of the foregoing or other known amino acids are suitablyemployed in this invention. Typically, amino acids are capable ofautocatalytically hydrolyzing the amino acid-steroid bond. Thus, theytypically contain, or upon being hydrolyzed in vivo, contain a freecarboxyl group or amine group.

Also of interest are hydrophobic amino acids such as mono- or di-alkylor aryl amino acids, cycloalkylamino acids and the like. These residues,together with R²⁹-R³⁴(R³¹-R³⁴ are defined below) can contribute to cellpermeability by modulating the lipophilicity of a formula 1 compound.Typically, the residue does not contain a sulfhydryl or guanidinosubstituent.

Peptide means one, 2, 3 or more of the two or more amino acids asdefined above are bonded together, usually by an amide bond. Variablegroups in the formula 1 compounds such as R¹-R¹⁰ can comprise a peptide.Typically the amino acids are linked through normal peptide bonds, e.g.,—CO—NH—, between adjacent amino acid residues. Peptides comprisedipeptides (dimers), tripeptides (trimers), short peptides of 4, 5, 6,8, 10 or 15 residues, and longer peptides or proteins having about 100or more residues. Formula 1 compounds that comprise a peptide can beused as immunogens, prodrugs or as synthetic precursors for othersteroid derivatives. In one embodiment, the peptide will contain apeptidolytic enzyme cleavage site at the peptide bond linking the firstresidue and the next residue distal to the steroid residue. Suchcleavage sites are optionally flanked by enzymatic recognitionstructures, e.g. particular residues recognized by a hydrolytic enzyme,e.g., a peptidase located in the serum or in cells.

Peptidolytic enzymes are well known, and in particular includecarboxypeptidases. Carboxypeptidases digest polypeptides by removingC-terminal residues, and are specific in many instances for particularC-terminal sequences. Such enzymes and their substrate requirements ingeneral are well known. For example, a dipeptide having a given pair ofresidues and a free carboxyl terminus is covalently bonded through itsα-amino group to the steroid nucleus. It is expected that the peptidewill be cleaved by the appropriate dipeptidase, protease or by chemicalhydrolysis, leaving the carboxyl of the proximal amino acid residue toautocatalytically cleave the amidate bond.

Examples of suitable dipeptidyl groups (designated by their singleletter symbols) are shown in the tables below.

SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr tyrosine G Gly glycine F Phephenylalanine M Met methionine A Ala alanine S Ser serine I Ileisoleucine L Leu leucine T Thr threonine V Val valine P Pro proline KLys lysine H His histidine Q Gln glutamine E Glu glutamic acid W Trptryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cyscysteine

Dipeptides AA, AR, AN, AD, AC, AE, AQ, AG, AH, AI, AL, AK, AM, AF, AP,AS, AT, AW, AY, AV, RA, RR, RN, RD, RC, RE, RQ, RG, RH, RI, RL, RK, RM,RF, RP, RS, RT, RW, RY, RV, NA, NR, NN, ND, NC, NE, NQ, NG, NH, NI, NL,NK, NM, NF, NP, NS, NT, NW, NY, NV, DA, DR, DN, DD, DC, DE, DQ, DG, DH,DI, DL, DK, DM, DF, DP, DS, DT, DW, DY, DV, CA, CR, CN, CD, CC, CE, CQ,CG, CH, CI, CL, CK, CM, CF, CP, CS, CT, CW, CY, CV, EA, ER, EN, ED, EC,EE, EQ, EG, EH, EI, EL, EK, EM, EF, EP, ES, ET, EW, EY, EV, QA, QR, QN,QD, QC, QE, QQ, QG, QH, QI, QL, QK, QM, QF, QP, QS, QT, QW, QY, QV, GA,GR, GN, GD, GC, GE, GQ, GG, GH, GI, GL, GK, GM, GF, GP, GS, GT, GW, GY,GV, HA, HR, HN, HD, HC, HE, HQ, HG, HH, HI, HL, HK, HM, HF, HP, HS, HT,HW, HY, HV, IA, IR, IN, ID, IC, IE, IQ, IG, IH, II, IL, IK, IM, IF, IP,IS, IT, IW, IY, IV, LA, LR, LN, LD, LC, LE, LQ, LG, LH, LI, LL, LK, LM,LF, LP, LS, LT, LW, LY, LV, KA, KR, KN, KD, KC, KE, KQ, KG, KH, KI, KL,KK, KM, KF, KP, KS, KT, KW, KY, KV, MA, MR, MN, MD, MC, ME, MQ, MG, MH,MI, ML, MK, MM, MF, MP, MS, MT, MW, MY, MV, FA, FR, FN, FD, FC, FE, FQ,FG, FH, FI, FL, FK, FM, FF, FP, FS, FT, FW, FY, FV, PA, PR, PN, PD, PC,PE, PQ, PG, PH, PI, PL, PK, PM, PF, PP, PS, PT, PW, PY, PV, SA, SR, SN,SD, SC, SE, SQ, SG, SH, SI, SL, SK, SM, SF, SP, SS, ST, SW, SY, SV, TA,TR, TN, TD, TC, TE, TQ, TG, TH, TI, TL, TK, TM, TF, TP, TS, TT, TW, TY,TV, WA, WR, WN, WD, WC, WE, WQ, WG, WH, WI, WL, WK, WM, WF, WP, WS, WT,WW, WY, WV, YA, YR, YN, YD, YC, YE, YQ, YG, YH, YI, YL, YK, YM, YF, YP,YS, YT, YW, YY, YV, VA, VR, VN, VD, VC, VE, VQ, VG, VH, VI, VL, VK, VM,VF, VP, VS, VT, VW, VY, VV

Such dipeptides include species where both amino acids are in the Lconfiguration, the D configuration or mixtures of configurations.

Tripeptides, i.e., 3 linked amino acid residues, are also usefulembodiments. Each amino acid in a tripeptide may be in an L, D or mixedconfiguration. Tripeptides include those where A, C, D, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W or Y is linked by a standard peptidebond to the amino or the carboxyl terminus of any of the dipeptideslisted above. The sequence —X1-pro-X2-(where X1 is any amino acid and X2is hydrogen, any amino acid residue or a carboxyl ester of proline) willbe cleaved by luminal carboxypeptidase to yield X1 with a free carboxyl,which in turn autocatalytically cleaves the amidate bond. X2 usuallywill be a benzyl ester of the carboxy group of X2. Other embodimentsinclude tetrapeptides such as ones where any two of the dipeptideslisted above, which may be the same or different dipeptides (e.g., AAand AA linked together or, e.g., AA and GI linked together), are linkedto each other by a peptide bond through the amino terminus or carboxylterminus. One, 2 or more tetrapeptides may bonded to the formula 1 orformula 2 compound through the tetrapeptide's amino or carboxylterminus.

In some embodiments, the formula 1 or formula 2 compound comprises oneor more amino acids or peptides having the structure (A), (B) or (C):

(A)R³²—NH—{[C(R²⁹)(R³⁰)]_(b)—C(O)—N(R³¹)}_(f)—[C(R²⁹)(R³⁰)]_(a)—C(O)—O-steroid,

(B)R³³—O—{C(O)—[C(R²⁹)(R³⁰)]_(d)—N(R³¹)}_(g)—C(O)—[C(R²⁹)(R³⁰)]_(c)—N(R³¹)—O-steroid,or

(C)R³³—O—{C(O)—[C(R²⁹)(R³⁰)]_(d)—N(R³¹)}_(e)—C(O)—[C(R²⁹)(R³⁰)]_(c)—N(R³¹)—C(O)—O-steroid,wherein

(A), (B) or (C) are independently selected and they are bonded to 1, 2,3 or more of R¹ through R⁴, where each R²⁹-R³¹ is independentlyselected; R²⁹ independently are —H or a C1-20 organic moiety (e.g., C₁₋₆alkyl, e.g. —CH₃ or —C₂H₅); R³⁰ independently are the side chain of anamino acid, including the side chain of naturally occurring amino acidsas described above, e.g., —H, —CH₃, —CH₂C₆H₅; R³¹ is —H or a protectinggroup; R³² and R³³ independently comprise —H, a protecting group, anester or an amide where each atom or group is independently chosen; a,b, c and d independently are 1, 2, 3, 4 or 5, usually 1; e, f and gindependently are an integer from 0 to about 1000, typically theyindependently are 0, 1, 2, 3, 4, 5, 6, 7 or 8; a, b, c and dindependently are 1 or 2; e, f and g independently are 0, 1, 2, 3, 4 or5.

If the amino acid(s) or residue(s) has 2 or more amine groups, e.g., alysinyl or arginyl, or ornithinyl residue, then R²⁹ is usually —H andR³⁰ may comprise —[C(R³⁴)₂]_(n2)N(R^(PR))— where n2 is 0, 1, 2, 3, 4, 5or 6, R^(PR) is —H or a protecting group and each R³⁴ independently is—H, C₁-C₂₀ optionally substituted alkyl, C₆-C₂₀ optionally substitutedaryl, C₇-C₂₀ optionally substituted alkylaryl, C₇-C₂₀ optionallysubstituted arylalkyl, C₁-C₂₀ optionally substituted alkoxy, C₆-C₂₀optionally substituted aryloxy or hydroxyl. Such compounds will containa plurality of steroid moieties. For example when both the epsilon (ε)or delta (δ) and alpha (α) amino groups of lysine or ornithine aresubstituted with steroid moieties the amidate is believed to be capableof releasing two molecules of active drug, each expected to emerge underdifferent pharmacokinetics and therefore further sustaining the drugrelease.

Salts of formula 1 compounds. Invention embodiments include salts andcomplexes of formula 1 compounds, including pharmaceutically acceptableor salts that are relatively non-toxic. Some of the formula 1 compoundshave one or more moieties that carry at least a partial positive ornegative charge in aqueous solutions, typically at a pH of about 4-10,that can participate in forming a salt, a complex, a composition withpartial salt and partial complex properties or other noncovalentinteractions, all of which we refer to as a “salt(s)”. Salts are usuallybiologically compatible or pharmaceutically acceptable or non-toxic,particularly for mammalian cells. Salts that are biologically toxic areoptionally used with synthetic intermediates of formula 1 compounds.When a water-soluble composition is desired, monovalent salts areusually used.

Metal salts typically are prepared by reacting the metal hydroxide witha compound of this invention. Examples of metal salts that areoptionally prepared in this way are salts containing Li⁺, Na⁺, and K⁺. Aless soluble metal salt can be precipitated from the solution of a moresoluble salt by adding a suitable metal compound. Invention salts may beformed from acid addition of certain organic acids, such as organiccarboxylic acids, and inorganic acids, such as alkylsulfonic acids orhydrogen halide acids, to acidic or basic centers on formula 1compounds, such as basic centers on the invention pyrimidine baseanalogs. Metal salts include ones containing Na⁺, Li⁺, K⁺, Ca⁺⁺ or Mg⁺⁺.Other metal salts may contain aluminum, barium, strontium, cadmium,bismuth, arsenic or zinc ion.

Salt(s) of formula 1 compounds may comprise a combination of appropriatecations such as alkali and alkaline earth metal ions or ammonium andquaternary ammonium ions with the acid anion moiety of the phosphoricacid or phosphonic acid group, which may be present in inventionpolymers or monomers.

Salts are produced by standard methods, including dissolving free basein an aqueous, aqueous-alcohol or aqueous-organic solution containingthe selected acid, optionally followed by evaporating the solution. Thefree base is reacted in an organic solution containing the acid, inwhich case the salt usually separates directly or one can concentratethe solution.

Suitable amine salts include amines having sufficient basicity to form astable salt, usually amines of low toxicity including trialkyl amines(tripropylamine, triethylamine, trimethylamine), procaine,dibenzylamine, N-benzyl-betaphenethylamine, ephenamine,N,N′-dibenzylethylenediamine, N-ethylpiperidine, benzylamine anddicyclohexylamine.

Salts include organic sulfonic acid or organic carboxylic acid salts,made for example by addition of the acids to basic centers, typicallyamines. Exemplary sulfonic acids include C₆₋₁₆ aryl sulfonic acids,C₆₋₁₆ heteroaryl sulfonic acids and C₁₋₁₆ alkyl sulfonic acids such asphenyl sulfonic acid, a-naphthalene sulfonic acid, β-naphthalenesulfonic acid, (S)-camphorsulfonic acid, methyl (CH₃SO₃H), ethyl(C₂H₆SO₃H), n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl,pentyl and hexyl sulfonic acids. Exemplary organic carboxylic acidsinclude C₁₋₁₆ alkyl, C₆₋₁₆ aryl carboxylic acids and C₄₋₁₆ heteroarylcarboxylic acids such as acetic, glycolic, lactic, pyruvic, malonic,glutaric, tartaric, citric, fumaric, succinic, malic, maleic, oxalic,hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic,salicylic, nicotinic and 2-phenoxybenzoic.

Invention salts include those made from inorganic acids, e.g., HF, HCl,HBr, HI, H₂SO₄, H₃PO₄, Na₂CO₃, K₂CO₃, CaCO₃, MgCO₃ and NaClO₃. Suitableanions, which are optionally present with a cation such a Ca⁺⁺, Mg⁺⁺,Li⁺⁺, Na⁺ or K⁺, include arsenate, arsenite formate, sorbate, chlorate,perchlorate, periodate, dichromate, glycodeoxycholate, cholate,deoxycholate, desoxycholate, taurocholate, taurodeoxycholate,taurolithocholate, tetraborate, nitrate, nitrite, sulfite, sulfamate,hyposulfite, bisulfite, metabisulfite, thiosulfate, thiocyanate,silicate, metasilicate, CN⁻, gluconate, gulcuronate, hippurate, picrate,hydrosulfite, hexafluorophosphate, hypochlorite, hypochlorate, borate,metaborate, tungstate and urate.

Salts also include the formula 1 compound salts with one or more aminoacids. Many amino acids are suitable, especially the naturally-occurringamino acids found as protein components, although the amino acidtypically is one bearing a side chain with a basic or acidic group,e.g., lysine, arginine, histidine or glutamic acid, or a neutral groupsuch as glycine, serine, threonine, alanine, isoleucine, or leucine.

The invention compositions include formula 1 compounds, their hydratesand the compounds in their un-ionized, as well as zwitterionic form.

Stereoisomers. The formula 1 compounds include enriched or resolvedoptical isomers at any or all asymmetric atoms as are apparent from thedepictions. Both racemic and diasteromeric mixtures, as well as theindividual optical isomers can be isolated or synthesized so as to besubstantially free of their enantiomeric or diastereomeric partners, andthese are all within the scope of the invention. Chiral centers may befound in formula 1 compounds at, for example, one or more of R¹, R², R³,R⁴, or R¹⁰.

One or more of the following methods are used to prepare theenantiomerically enriched or pure isomers herein. The methods are listedin approximately their order of preference, i.e., one ordinarily shouldemploy stereospecific synthesis from chiral precursors beforechromatographic resolution before spontaneous crystallization.

Stereospecific synthesis is described in the examples. Methods of thistype conveniently are used when the appropriate chiral starting materialis available and reaction steps are chosen do not result in undesiredracemization at chiral sites. One advantage of stereospecific synthesisis that it does not produce undesired enantiomers that must be removedfrom the final product, thereby lowering overall synthetic yield. Ingeneral, those skilled in the art would understand what startingmaterials and reaction conditions should be used to obtain the desiredenantiomerically enriched or pure isomers by stereospecific synthesis.

If a suitable stereospecific synthesis cannot be empirically designed ordetermined with routine experimentation then those skilled in the artwould turn to other methods. One method of general utility ischromatographic resolution of enantiomers on chiral chromatographyresins. These resins are packed in columns, commonly called Pirklecolumns, and are commercially available. The columns contain a chiralstationary phase. The racemate is placed in solution and loaded onto thecolumn, and thereafter separated by HPLC. See for example, ProceedingsChromatographic Society—International Symposium on Chiral Separations,Sep. 3-4, 1987. Examples of chiral columns that could be used to screenfor the optimal separation technique would include Diacel Chriacel OD,Regis Pirkle Covalent D-phenylglycine, Regis Pirkle Type 1A, AstecCyclobond II, Astec Cyclobond III, Serva Chiral D-DL=Daltosil 100,Bakerbond DNBLeu, Sumipax OA-1000, Merck Cellulose Triacetate column,Astec Cyclobond I-Beta, or Regis Pirkle Covalent D-Naphthylalanine. Notall of these columns are likely to be effective with every racemicmixture. However, those skilled in the art understand that a certainamount of routine screening may be required to identify the mosteffective stationary phase. When using such columns it is desirable toemploy embodiments of the compounds of this invention in which thecharges are not neutralized, e.g., where acidic functionalities such ascarboxyl are not esterified or amidated.

Another method entails converting the enantiomers in the mixture todiasteriomers with chiral auxiliaries and then separating the conjugatesby ordinary column chromatography. This is a very suitable method,particularly when the embodiment contains free carboxyl, amino orhydroxyl that will form a salt or covalent bond to a chiral auxiliary.Chirally pure amino acids, organic acids or organosulfonic acids are allworthwhile exploring as chiral auxiliaries, all of which are well knownin the art. Salts with such auxiliaries can be formed, or they can becovalently (but reversibly) bonded to the functional group. For example,pure D or L amino acids can be used to amidate the carboxyl group ofinvention embodiments that comprise a carboxyl group and then separatedby chromatography.

Enzymatic resolution is another method of potential value. In suchmethods one prepares covalent derivatives of the enantiomers in theracemic mixture, generally lower alkyl esters (for example of carboxyl),and then exposes the derivative to enzymatic cleavage, generallyhydrolysis. For this method to be successful an enzyme must be chosenthat is capable of stereospecific cleavage, so it is frequentlynecessary to routinely screen several enzymes. If esters are to becleaved, then one selects a group of esterases, phosphatases, andlipases and determines their activity on the derivative. Typicalesterases are from liver, pancreas or other animal organs, and includeporcine liver esterase.

If the enantiomeric mixture separates from solution or a melt as aconglomerate, i.e., a mixture of enantiomerically pure crystals, thenthe crystals can be mechanically separated, thereby producing theenantiomerically enriched preparation. This method, however, is notpractical for large-scale preparations and is of limited value for trueracemic compounds.

Asymmetric synthesis is another technique for achieving enantiomericenrichment. For example, a chiral protecting group is reacted with thegroup to be protected and the reaction mixture allowed to equilibrate.If the reaction is enantiomerically specific then the product will beenriched in that enantiomer.

Further guidance in the separation of enantiomeric mixtures can befound, by way of example and not limitation, in “Enantiomers, Racemates,and resolutions”, Jean Jacques, Andre Collet, and Samuel H. Wilen(Krieger Publishing Company, Malabar, Fla., 1991, ISBN 0-89464-618-4):Part 2, Resolution of Enantiomer Mixture, pages 217-435; moreparticularly, section 4, Resolution by Direct Crystallization, pages217-251, section 5, Formation and Separation of Diastereomers, pages251-369, section 6, Crystallization-Induced Asymmetric Transformations,pages 369-378, and section 7, Experimental Aspects and Art ofResolutions, pages 378-435; still more particularly, section 5.1.4,Resolution of Alcohols, Transformation of Alcohols into Salt-FormingDerivatives, pages 263-266, section 5.2.3, Covalent Derivatives ofAlcohols, Thiols, and Phenols, pages 332-335, section 5.1.1, Resolutionof Acids, pages 257-259, section 5.1.2, Resolution of Bases, pages259-260, section 5.1.3, Resolution of Amino Acids, page 261-263, section5.2.1, Covalent Derivatives of Acids, page 329, section 5.2.2, Covalentderivatives of Amines, pages 330-331, section 5.2.4, CovalentDerivatives of Aldehydes, Ketones, and Sulfoxides, pages 335-339, andsection 5.2.7, Chromatographic Behavior of Covalent Diastereomers, pages348-354.

General methods. Methods have been described, for example Karl Fischer(KF) and loss on drying (LOD), to determine the content of water orsolvents in various compositions. LOD measures all volatiles in asample, while KF is typically used to measure all water. When water isthe only volatile present, LOD values are equal to or less than KFvalues for a given composition. KF measures water in hydrates of acompound and LOD determines both water and the amount of other volatilesthat may be present. Invention compositions and formulations areconveniently assayed for water content by KF titration (e.g., using aMetrohm 684 KF Coulometer or equivalent) according to a publishedprocedure (U.S. Pharmacopoeia, vol. 23, 1995, chapter <921>, U.S.Pharmacopeial Convention, Inc., Rockville, Md.) and manufacturer'sCoulometer instructions. The amount of material used in the assay, about50-100 mg, is measured using a five-place analytical balance (Sartorius,Model RC210D, or a suitable equivalent). The amounts of water specifiedin invention compositions and formulations are the amount obtained by KFanalysis.

Powder X-ray diffraction (XRD) methods have been used to characterizevarious crystalline compounds (see, e.g., U.S. Pharmacopoeia, volume 23,1995, <941>, p. 1843-1845, U.S. Pharmacopeial Convention, Inc.,Rockville, Md.; Stout et al., X-Ray Structure Determination; A PracticalGuide, MacMillan Co., New York, N.Y., 1986). The diffraction pattern, orportions thereof, obtained from a crystalline compound is usuallydiagnostic for a given crystal form, although weak or very weakdiffraction peaks may not always appear in replicate diffractionpatterns obtained from successive batches of crystals. Also, therelative intensities of XRD bands, particularly at low angle X-rayincidence values (low Theta), may vary due to preferred orientationeffects arising from differences in, e.g., crystal habit, particle sizeor other measurement conditions. Peaks on XRD spectra are typicallydefined at a given Theta value +/−about 0.1 to 0.2. XRD information fromthe 1, 2, 3, 4, 5 or more main intensity XRD peaks, optionally combinedwith one or more other diagnostic data (melting point, DSC, IR), isusually suitable to characterize or describe a crystalline material suchas 16α-bromoepiandrosterone hemihydrate (“BrEA hemihydrate”) from othercrystal forms that contain the same compound.

Other techniques that are used to identify or describe a crystallinematerial such as BrEA hemihydrate include melting point (MP),differential scanning calorimetry (DSC) and infrared absorptionspectroscopy (IR) data. DSC measures thermal transition temperatures atwhich a crystal absorbs or releases heat when its crystal structurechanges or it melts. MP data and DSC thermal transition temperatures aretypically reproducible within about 1 or 2° C. on successive analyses.IR measures absorption of infrared light that is associated with thepresence of particular chemical bonds that are associated with groups,e.g., hydroxyl, that vibrate in response to particular lightwavelengths.

BrEA hemihydrate. Formula 1 compounds include BrEA hemihydrate

which is optionally characterized by reference to one or more physicalproperties such as its melting point, infrared absorption spectrum orits powder X-ray diffraction spectrum. Related embodiments include BrEAhemihydrate and one or more excipients, e.g., suitable for humanpharmaceutical use or for veterinary use. Another related embodiment isa method to make BrEA hemihydrate comprising precipitating orcrystallizing BrEA from a solution comprising ethanol and water.

BrEA hemihydrate is typically substantially free of other forms of BrEA,such as amorphous BrEA or anhydrous BrEA. As used herein, BrEAhemihydrate or crystalline BrEA hemihydrate refers to solid BrEA andwater having an ordered arrangement of substantially all of theconstituent molecules in a defined three-dimensional spatial pattern orlattice. Crystalline BrEA hemihydrate may comprise one or more crystalhabits, e.g., tablets, rods, plates or needles. BrEA hemihydrate that issubstantially free of other forms of BrEA means a dry or substantiallydry (where a liquid(s) comprises less than about 10% w/w of the totalweight) solid preparation where more than about 55% w/w of the BrEA inthe preparation is present as BrEA hemihydrate. Such compositionstypically comprise at least about 60% w/w, or at least about 70% w/w, orat least about 80% w/w, usually at least about 90% w/w or at least about95% w/w, or at least about 98% w/w of BrEA hemihydrate, with theremaining BrEA being present as other forms of BrEA such as theamorphous or anhydrous BrEA. Solid BrEA hemihydrate will typicallycomprise at least about 90% w/w, usually at least about 97% or about 98%w/w of the compound and less than about 10% w/w, usually less than about3% or 2% w/w of by-products, which may include the 16β isomer of BrEA orone or more by-products of BrEA synthesis. Often the amount of solidBrEA that is present in a solid or a liquid medium will not containdetectable amounts of other forms of BrEA (using standard analyticalmethods such as, e.g., FTIR, DSC or XRD) and the hemihydrate will maythus comprise about 99-100% w/w of the total amount of BrEA that ispresent.

Other invention embodiments include compositions that comprise asubstantial amount of BrEA hemihydrate that is present in compositionsthat comprise one or more other forms of BrEA, e.g., amorphous BrEA oranhydrous BrEA, and optionally one or more additional components, suchas any excipient described herein. As used herein, the “substantialamount” of BrEA hemihydrate in these compositions comprises at leastabout 15-20% w/w or at least about 20% w/w of BrEA hemihydrate of thetotal amount of BrEA that is present, typically at least about 25% w/w,more typically at least about 30% w/w, often at least about 35% w/w andusually at least about 45% w/w. These compositions are generally solids,e.g., formulations or unit dosages, but they also include suspensions,precipitates, gels and colloids that contain solid BrEA. Suchsuspensions or precipitates may arise from, e.g., precipitation of BrEAhemihydrate from a solution that contains water or from addition ofsolid BrEA to a liquid excipient(s). Obviously, compositions thatcomprise a substantial amount of BrEA may be substantially free of otherforms of solid BrEA as discussed above.

BrEA hemihydrate may conveniently be identified by reference to BrEAhemihydrate characterized by one or more of (1) its melting ordecomposition point or range (optionally expressed as +/−2° C.), (2) oneor more BrEA hemihydrate DSC transition temperatures or ranges (any ofwhich may be optionally expressed as +/−2° C.), (3) one or morecharacteristic BrEA hemihydrate IR absorption bands, (4) 1, 2, 3, 4, 5,6 or more of the highest intensity XRD peaks (any one or more of whichare optionally expressed as +/−0.1° Theta or +/−0.2° Theta) obtainedfrom an XRD spectrum of BrEA hemihydrate using Cu—Kα radiation (e.g.,obtained essentially according to the method described at U.S.Pharmacopoeia, volume 23, 1995, <941>, p. 1843-1845), (5) the presenceof less than about 3% or less than about 2% w/w of other compounds, (6)a water content of dry BrEA hemihydrate of about 2.5% w/w (e.g.,2.3-2.7% w/w), where dry BrEA hemihydrate means compound dried byfiltration, optionally washed once with an anhydrous solvent such ashexane, filtered again and dried in vacuo at about 60° C. until nofurther weight loss occurs over 24 hours at about 60° C. (e.g., wherewater content is determined essentially by the Karl Fisher or othermethod described at U.S. Pharmacopoeia, vol. 23, 1995, p 1801-1802 or1840-1843 methods <731> or <921>), (7) cell constants and theorientation matrix obtained from single crystal X-ray crystallography ofBrEA hemihydrate (obtained, e.g., essentially as described in WO99/04774 at example 13), (8) a description of crystal shapes as observedat about 100× magnification to about 150× magnification by polarizedlight microscopy or (9) average BrEA hemihydrate crystal size and shapedescriptions.

Thus, for example, BrEA hemihydrate may be characterized by or one ormore of its IR absorption bands, e.g., the carbonyl peaks at 1741 cm⁻¹and 1752 cm⁻¹, and either its melting or decomposition point or rangeand/or 1, 2, 3, 4, 5, 6 or more of the XRD peaks (usually the highestintensity peaks) at Theta (X-ray diffraction angle) values of 17.8,23.8, 24.2, 26.9-27.2, 28.6, 30.1 and 32.2.

BrEA hemihydrate is suitable to prepare compositions comprising anexcipient(s) suitable for human pharmaceutical use or for veterinaryuse. Such compositions are used to prepare formulations and unitdosages. Unit dosages typically comprise tablets, capsules, lozenges orsterile solutions, including sterile solutions for parenteraladministration. Solid unit dosage forms typically comprise about 1-1000mg of BrEA hemihydrate, typically about 20-400 mg, e.g., about 10 mg,about 25 mg, about 50 mg, about 100 mg, about 150 mg or about 250 mg perunit dose.

The invention provides a method to make BrEA hemihydrate comprisingcontacting water, 16α-bromo-3β-hydroxy-5α-androstan-17-one and a C1-C6alcohol (e.g., methanol, ethanol, propanol, isopropanol, butanol) andwater. Typically the only one C1-C6 alcohol is present, e.g., ethanol,which is anhydrous or which may comprise up to about 2% w/w water. Insome embodiments, the method utilizes a solution that comprises about5-25% w/w water, about 30-45% w/w ethanol and about 30-45% w/w of a BrEApreparation. Typical BrEA preparations are solid preparations thatcomprise at least about 80% w/w, usually at least about 90% w/w or atleast about 95% w/w of BrEA. The solutions may comprise about 18-22% w/wwater, about 37-43% w/w ethanol and about 37-43% w/w of a BrEApreparation. In conducting the precipitation or crystallization method,the solution will typically be at a temperature of about −20° C. toabout 45° C., usually at about 0° C. to about 20° C. The solution ismaintained at this temperature range for about 30 minutes to about 12hours and the solution is optionally agitated using slow to moderateagitation during crystallization.

A related embodiment comprises a method to prepare BrEA hemihydratecomprising precipitating BrEA from a solution comprising at least about15-25% w/w water, about 35-45% w/w of a BrEA preparation and at leastabout 35-45% w/w of one or more water-miscible solvents, typically C₁₋₆alcohols (methanol, ethanol, propanol, isopropanol, butanol). The BrEApreparation may optionally comprise one or more by-products of BrEAsynthesis. Typical BrEA hemihydrate preparations or batches compriseless than about 5% w/w, usually less than about 3% or about 2% w/w ofother compounds, such as by-products of BrEA synthesis. Aspects of thismethod include contacting water with an organic solution that comprisesBrEA and an organic solvent such as a C1-C6 alcohol (e.g., ethanol) oracetone. Addition of water to such solutions leads to precipitation ofBrEA hemihydrate. Solutions that contain BrEA hemihydrate crystals orprecipitate are invention embodiments that are used to prepare solidBrEA that is later dried and stored, typically at ambient temperaturesand typically under conditions that limits or blocks light that reachesthe compound.

Precipitation or crystallization of BrEA hemihydrate fromwater-containing solutions is accomplished by known methods, e.g.,reducing the solution's temperature, using saturated or nearly saturatedBrEA solutions, vacuum concentration of saturated or nearly saturatedBrEA solutions (which is typically conducted at a relatively lowtemperature, usually about 15-25° C.), seeding with saturated or nearlysaturated BrEA solutions with BrEA hemihydrate crystals (e.g., about10-100 mg per 1-10 L of solution), by heating a saturated or nearlysaturated BrEA solution (about 25-35° C. for a few minutes followed byallowing the temperature to fall or by actively cooling the solution)and optionally seeding the solution with BrEA hemihydrate crystals or byaddition of a liquid, e.g., additional water or ethanol, to a saturatedor nearly saturated BrEA ethanol-water solution, which causes thesolution to become supersaturated. BrEA may also be precipitated fromother solvents or solvent systems, including acetone andacetone-ethanol. Such solvents are typically water miscible. Two-stageprecipitation of BrEA may also be used to recover solid BrEAhemihydrate, e.g., initial precipitation and recovery of the solid,followed by either cooling and seeding of the mother liquor or byallowing the mother liquor to stand, e.g., for about one, two or moredays at ambient temperature, to obtain a second crop of crystals. Also,BrEA hemihydrate crystals may optionally be recrystallized, essentiallyas described herein, to further increase the purity of the final solid.Methods for crystallizing organic compounds have been described, e.g.,A. S. Myerson, Handbook of Industrial Crystallization, 1993,Butterworth-Heinemann, Stoneham, Mass., p 1-101.

Other related embodiments comprise a product produced by the process ofcontacting a solution comprising BrEA and an organic solvent with water.Typically the solutions are as described above, e.g., a solutioncomprising about 3-5% v/v water and at least about 40% v/v of one ormore water-miscible solvents, typically polar solvents such as C₁₋₆alcohols or ketones (e.g., methanol, ethanol, propanol, isopropanol,butanol, typically ethanol or acetone). Such processes are accomplishedby any one or more of the techniques described in the paragraph above,e.g., cooling of a saturated or nearly saturated BrEA water-ethanolsolution and optionally seeding the cooled solution with BrEAhemihydrate. An embodiment related to this comprises solutions or solidsthat comprise wet BrEA hemihydrate crystals or wet filtered orcentrifuged BrEA hemihydrate cakes, which may be obtained aftercrystallization. Examples of these embodiments include adding water to aBrEA-alcohol solution, e.g., slow addition of about 0.5-1.5 volumes orabout 0.8-1.2 volumes of water to about 6 volumes of a BrEA-ethanolsolution to obtain BrEA hemihydrate. Other examples of these embodimentsinclude adding water to a BrEA-ketone solvent solution, e.g., slowaddition of about 0.5-1.5 volumes or about 0.8-1.2 volumes of water toabout 10 volumes of a BrEA-acetone solution to obtain BrEA hemihydrate.

Another related embodiment is BrEA hemihydrate that is milled to anaverage particle size of about 0.01-200 μM, or about 0.1-10 μM or about0.5-5 μM. Average particle size or diameter for milled BrEA hemihydratemay thus be relatively small, e.g., about 0.03-2.0 μM or about 0.1-1.0μM, or somewhat larger, e.g., about 0.5-5.0 μM or about 1-5.0 μM. MilledBrEA hemihydrate is suitable for preparing solid formulations andparenteral formulations for human or veterinary use. The milled materialfacilitates dissolution of BrEA hemihydrate in solvents or excipientsand facilitates mixing with solids or solid excipients.

While it is possible to administer BrEA hemihydrate as a pure compoundto a subject, it is usually presented as a solid formulation or used toprepare a liquid formulation. Formulations will typically be used toprepare unit dosages, e.g., tablets, capsules or lozenges for oral,buccal or sublingual administration, that comprise about 10-1000 mg ortypically about 25-400 mg of BrEA hemihydrate. Alternatively,embodiments include a product for parenteral (e.g., subcutaneous,subdermal, intravenous, intramuscular, intraperitoneal) administrationmade by the process of contacting BrEA hemihydrate and a liquidexcipient, e.g., any one, two, three or more of PEG 100, PEG 200, PEG300, PEG 400, propylene glycol, benzyl benzoate, benzyl alcohol orethanol, and optionally sterilizing the solution and optionallydispensing the solution into vials or ampules (typically amber glass),which may be single-use or multi-use and optionally storing theformulation at reduced temperature (about 0-12° C., or about 2-10° C.).Such products for parenteral administration typically comprise BrEA at aconcentration of about 10-170 mg/mL, usually at about 20-110 mg/mL orabout 30-100 mg/mL, and optionally one or more of a salt, buffer orbacteriostat or preservative (e.g., NaCl, BHA, BHT or EDTA).

Other embodiments include a product produced by the process ofcontacting BrEA hemihydrate, which may be substantially free of otherforms of BrEA, with an excipient suitable for human pharmaceutical useor for veterinary use. The product is useful to make formulations orunit dosage forms that contain the hemihydrate. Exemplary excipientsinclude one or more of those disclosed herein, e.g., sucrose, mannitol,starch, carboxymethyl cellulose, magnesium stearate and the like.

Specific embodiments of formula 1 compounds. Other embodiments includecompounds, compositions and formulations where one or more variablegroups that are bonded to the formula 1 compounds, e.g., one or more ofR¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸ comprise an amino acid or a peptide, e.g.,R¹, R² or R⁴ comprises an amino acid or a peptide, R³ is a halogen andR⁵ and R⁶ are both —CH₃. The peptide at one or more of R¹-R⁶ cancomprise a cell surface binding peptide such as the entire protein or asequence from fibronectin or retronectin.

In the formula 1 compounds, each R⁴ is independently selected. In someembodiments one R⁴ is hydrogen and the other is another moiety. In otherembodiments, both R⁴ are independently selected moieties other thanhydrogen, e.g., a C1 to C20 organic moiety.

R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸ include moieties, e.g., esters, thioesters,thionoesters, carbonates, amino acids, peptides and/or carbamates, thatare chemically and/or enzymatically hydrolyzable, often underphysiological conditions. Such moieties are independently chosen.Typically these moieties will give rise to —OH, —SH or —NH₂ at the R¹-R⁶positions of the steroid nucleus. Embodiments of formula 1 compoundsinclude ones where (1) one of R¹, R² and R⁴ is a hydrolyzable moiety(e.g., ester, thioester, thionoester, carbonate, amino acid, peptide orcarbamate), the other two of R¹, R² and R⁴ are —H, R³ is not hydrogenand R⁵ and R⁶ are both —CH₃, (2) two of R¹, R² and R⁴ are hydrolyzablemoieties (e.g., independently chosen esters, thioesters, thionoesters,carbonates, amino acids, peptides and/or carbamates), the other of R¹,R² and R⁴ is —H, R³ is not hydrogen and R⁵ and R⁶ are both —CH₃, (3) R¹,R² and R⁴ are hydrolyzable moieties, R³ is not hydrogen and R⁵ and R⁶are both —CH₃. In these embodiments, the R³ group is typically in theβ-configuration and the R¹, R² and R⁴-R⁶ groups are typically in theα-configuration.

In other embodiments, one or more of R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸,usually one, comprises an amino acid or a peptide, while the remaininggroups are independently selected from the moieties defined herein. Inthese embodiments, the peptides are typically dimers (dipeptides) ortrimers (tripeptides). For example one of R¹, R² or R⁴ comprises anamino acid, the remaining of R¹, R² or R⁴ independently comprise —OH,═O, an ester, a carbonate or a carbamate, while R³ is a halogen,hydroxyl or an ester and R⁵ and R⁶ independently are —H, —(CH₂)_(n)—CH₃,—(CH₂)_(n)—CH₂OH, or —(CH₂)_(n)—CH₂F, —(CH₂)₂₋₄—O—(CH₂)₂₋₄—CH₃, where nis 0, 1, 2, 3, 4, 5, 6, 7 or 8 often 0, 1, or 2, usually 0. Typicallythe ester, carbonate or carbamate are hydrolyzable under physiologicalconditions.

Hydrolyzable moieties typically comprise acyl groups, esters, ethers,thioethers, amides, amino acids, peptides, carbonates and/or carbamates.In general, the structure of hydrolyzable moieties is not critical andcan vary. In some embodiments, these moieties contain a total of about 4to about 10 carbon atoms. These hydrolyzable moieties in otherembodiments comprise an organic moiety, as described above for ester,that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15 or 16 carbonatoms and 1, 2, 3, 4, 5, 6, 7 or 8 heteroatoms, e.g., oxygen, nitrogenor sulfur. These hydrolyzable moieties can comprise no groups that arecharged in plasma, blood, intracellular cytoplasm or in the gut, or theycan comprise 1, 2, 3 or more positive, negative or positive and negativecharges under one or more of these conditions. The charges may befractional depending on the group and the conditions it is under. Thesehydrolyzable moieties may comprise 1, 2, 3, 4 or more substitutions at ahydrogen atom(s) and/or a carbon atom(s), e.g., —OH, protected hydroxyl,—SH, protected thiol, carboxyl, protected carboxyl, amine, protectedamine, —O—, —S—, —CO—, —CS—, alkoxy, alkylthio, alkenyloxy, aryl,—OP(O)(O)—O—, —OS(O)(O)—O— and/or heterocycle. Such substitutions areindependently selected. Embodiments of formula 1 compounds include oneswherein one, two, three, four or more of the variable groups that arebonded to the steroid rings, e.g., R¹-R⁶ or R¹⁰, comprise a moiety thatcan hydrolyze or metabolize to, e.g., a —H, —OH, ═O, —SH, ═S, —COOH,—NH₂, —CH₂OH, —CH₂SH, —C(O)—C1-C6 alkyl-OH, —C(O)—C1-C6 alkyl-SH,—C(S)—C1-C6 alkyl-OH, —C(O)—C1-C6 alkyl or —C(O)—NH₂ atom or group.

Formula 1 compounds that comprise a hydrolyzable moiety(ies) may includeone or more independently chosen —O—CHR²⁴C(O)OR²⁵, —S—CHR²⁴C(O)OR²⁵,—NH—CHR²⁴C(O)OR²⁵, —O—CHR²⁴C(S)OR²⁵, —S—CHR²⁴C(S)OR²⁵,—NH—CHR²⁴C(S)OR²⁵, —O—CHR²⁴OC(O)R²⁵, —S—CHR²⁴OC(O)R²⁵,—NH—CHR²⁴OC(O)R²⁵, —O—CHR²⁵, —O—CHR²⁴C(O)N(R²⁵)₂, —NH—CHR²⁴C(O)N(R²⁵)₂,—O—CHR²⁴OR²⁵, —S—CHR²⁴OR²⁵, —NH—CHR²⁴OR²⁵, —O—CHR²⁴C(R²⁵)₂CH₂OX,—S—CHR²⁴C(R²⁵)₂CH₂OX, —NH—CHR²⁴C(R²⁵)₂CH₂OX, —O—CHR²⁴C(R²⁵)₂OX,—S—CHR²⁴C(R²⁵)₂OX or —NH—CHR²⁴C(R²⁵)₂OX, groups that one or more ofR¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸ comprise. For these hydrolyzable moieties,R²⁴ independently is —H, —CH₂—C₆H₅, —CH₂CH₂—C₆H₅, C₁₋₈ alkyl, C₂₋₈alkenyl, aryl or heterocycle where each alkyl, alkenyl, aryl andheterocycle moiety is independently optionally substituted with 1, 2, or3, usually 1, —O—, —S—, —NH—, halogen, aryl, —OX, —SX, —NHX, ketone (═O)or —CN moieties or the C₁₋₈ alkyl is optionally substituted with 3, 4, 5or 6 halogens, and X is —H or a protecting group. Exemplary R²⁴ are —H,—CH₃, —C₂H₅, —CH₂—C₁₋₅ optionally substituted alkyl, —CH₂CH₂—C₁₋₄optionally substituted alkyl and —CH₂CH₂—O—C₁₋₄ optionally substitutedalkyl. R²⁵ independently is —H or a C₁₋₃₀ organic moiety such as—CH₂—C₆H₅, —CH₂CH₂—C₆H₅, C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, C₂₋₁₂ alkynyl,aryl, a heterocycle, —CH₂-heterocycle or —CH₂-aryl, where each alkyl,alkenyl, alkynyl, aryl, heterocycle, —CH₂-heterocycle or —CH₂-arylmoiety is independently optionally substituted with 1 or 2, usually 1,—O—, —S—, —NH—, halogen, aryl, —OX, —SX, —NHX, ketone (═O), —C(O)OX or—CN moieties or the C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl or aryl, are optionallyindependently substituted with 3, 4, 5 or 6 halogens, where X is —H or aprotecting group, or the aryl, heterocycle, —CH₂-heterocycle or—CH₂-aryl moieties are optionally independently substituted with 1, 2 or3 C₁₋₄ alkyl moieties or with 1, 2 or 3 C₁₋₄ alkoxy moieties at the arylmoiety or at the heterocycle, usually at a ring carbon. Exemplary R²⁵are —H, —CH₃, —C₂H₅, —C₃H₇, —C₄H₉, —C₆H₁₃, —C₆H₅, —C₆H₄OH, —C₆H₄OCH₃,—C₆H₄F, —CH₂—C₁₋₅ optionally substituted alkyl, —CH₂CH₂—(S)₀₋₁—C₁₋₄optionally substituted alkyl and —CH₂CH₂—O—C₁₋₄ optionally substitutedalkyl.

Invention embodiments include a composition comprising (1) a compound offormula 1 or 2 and one or more nonaqueous liquid excipients, wherein thecomposition comprises less than about 3% v/v water and (2) a compound offormula 1 or 2 and one or more solid excipients.

Invention embodiments include one or more compounds of formula 1 orformula 2 including formula 1 compounds having the structure

wherein,

R¹, R², R³, R⁴, R⁵, R⁶ and R¹⁰ independently are —H, —OR^(PR), —SR^(PR),—N(R^(PR))₂, —O—Si—(R¹³)₃, —CHO, —CHS, —SCN, —CH═NH, —CN, —NO₂, —OSO₃H,—OPO₃H, an ester, a thioester, a thionoester, a phosphoester, aphosphothioester, a phosphonoester, a phosphiniester, a sulfite ester, asulfate ester, an amide, an amino acid, a peptide, an ether, athioether, an acyl group, a thioacyl group, a carbonate, a carbamate, anacetal, a thioacetal, a halogen, an optionally substituted alkyl group,an optionally substituted alkenyl group, an optionally substitutedalkynyl group, an optionally substituted aryl moiety, an optionallysubstituted heteroaryl moiety, an optionally substituted monosaccharide,an optionally substituted oligosaccharide, a nucleoside, a nucleotide,an oligonucleotide, a polymer, or,

one, two or more of R¹, R², R³, R⁴, R⁵, R⁶ and R¹⁰ independently are ═O,═S, ═N—OH, ═CH₂ or a spiro ring and the hydrogen atom that is bonded tothe same carbon atom is absent, or,

R³ and R⁴ together comprise a structure of formula 2

R⁷ is —C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—C(R¹⁰)₂—,—C(R¹⁰)₂—O—C(R¹⁰)₂—, —C(R¹⁰)₂—S—C(R¹⁰)₂—, —C(R¹⁰)₂—NR^(PR)—C(R¹⁰)₂—,—O—, —O—C(R¹⁰)₂—, —S—, —S—C(R¹⁰)₂—, —NR^(PR)—, —NR^(PR)—C(R¹⁰)₂—,—CHR¹⁰—, —CHR¹⁰—CHR¹⁰—, —CHR¹⁰—CHR¹⁰—CHR¹⁰—, —CHR¹⁰—O—CHR¹⁰—,—CHR¹⁰—S—CHR¹⁰—, —CHR¹⁰—NR^(PR)—CHR¹⁰—, —O—, —O—CHR¹⁰—, —S—, —S—CHR¹⁰—,—NR^(PR)— or —NR^(PR)—CHR¹⁰—;

R⁸ and R⁹ independently are —C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—, —O—,—O—C(R¹⁰)₂—, —S—, —S—C(R¹⁰)₂—, —NR^(PR)—, —NR^(PR)—C(R¹⁰)₂—, —CHR¹⁰—,—CHR¹⁰—CHR¹⁰—, —O—, —O—CHR¹⁰, —S—, —S—CHR¹⁰—, —NR^(PR)— or—NR^(PR)—CHR¹⁰—, or one or both of R⁸ or R⁹ independently are absent,leaving a 5-membered ring;

R⁸ and R⁹ independently are —CHR¹⁰—, —CHR¹⁰—CHR¹⁰—, —O—, —O—CHR¹⁰—, —S—,—S—CHR¹⁰—, —NR^(PR)— or —NR^(PR)—CHR¹⁰—, or R⁸ or R⁹ independently isabsent, leaving a 5-membered ring;

R¹³ independently is C₁₋₆ alkyl;

R^(PR) independently is —H or a protecting group for, e.g., an O, N or Satom;

D is a heterocycle or a 4-, 5-, 6- or 7-membered ring that comprisessaturated carbon atoms, wherein 1, 2 or 3 ring carbon atoms of the 4-,5-, 6- or 7-membered ring are optionally independently substituted with—O—, —S— or —NR^(PR)— or where 1, 2 or 3 hydrogen atoms of theheterocycle or where 1, 2 or 3 hydrogen atoms of the 4-, 5-, 6- or7-membered ring are substituted with —OR^(PR), —SR^(PR), —N(R^(PR))₂,—O—Si—(R¹³)₃, —CHO, —CHS, —CH═NH, —CN, —NO₂, —OSO₃H, —OPO₃H, an ester, athioester, a thionoester, a phosphoester, a phosphothioester, aphosphonoester, a phosphiniester, a sulfite ester, a sulfate ester, anamide, an amino acid, a peptide, an ether, a thioether, an acyl group, athioacyl group, a carbonate, a carbamate, an acetal, a thioacetal, ahalogen, an optionally substituted alkyl group, an optionallysubstituted alkenyl group, an optionally substituted alkynyl group, anoptionally substituted aryl moiety, an optionally substituted heteroarylmoiety, an optionally substituted monosaccharide, an optionallysubstituted oligosaccharide, a nucleoside, a nucleotide, anoligonucleotide or a polymer, or,

one or more of the ring carbons are substituted with independentlyselected ═O, ═S, ═N—OH, ═CH₂ or a spiro ring, or

D comprises two 5- or 6-membered rings, wherein the rings are fused orare linked by 1 or 2 bonds, wherein one, two or three of R⁷, R⁸ and R⁹are not —CHR¹⁰— or —C(R¹⁰)₂—.

In the formula 1 or formula 2 compounds, whenever a variable moiety suchas R⁷, R⁸ or R⁹ is defined to include moieties such as —O—CHR¹⁰— or—NR^(PR)—CHR¹⁰—, it is intended that such moieties can be present ineither orientation relative to the other ring atoms that may be present,i.e., —O—CHR¹⁰—, —NR^(PR)—CHR¹⁰—, —CHR¹⁰—O— and —CHR¹⁰—NR^(PR)— are allincluded.

Embodiments of formula 1 compounds include or exclude any subset ofcompounds within the definition of formula 1, provided that at least onecompound remains. For example, a subset of formula 1 compounds that aremay be included, for example in the invention nonaqueous formulationsand in the invention intermittent dosing protocols and immune modulationmethods, are formula 1 compounds where R² is hydroxyl, or a group thatcan hydrolyze or metabolize to hydroxyl or thiol, in eitherconfiguration and R⁵ and R⁶ are methyl in the α-configuration. A subsetcompounds that are optionally excluded from formula 1 compoundscomprises one or all compounds that are disclosed in one or more priorart references or publications, e.g., one or more compounds that aredisclosed in one or more of the references cited herein, especially forthose compounds that can render any claim or embodiment unpatentable fornovelty, obviousness and/or inventive step reasons.

Exemplary embodiments of species and genera of formula 1 compounds arenamed as described below.

Group 1. Exemplary embodiments include the formula 1 compounds namedaccording to the compound structure designations given in Tables A and Bbelow. Each compound named in Table B is depicted as a compound havingformula B

where R⁵ and R⁶ are both —CH₃, there is no double bond at the 1-2-, 4-5-or 5-6-positions, one R⁴ is hydrogen, R⁷, R⁸ and R⁹ are all —CH₂— andR¹, R², R³ and R⁴ are the substituents designated in Table A. Thecompounds named according to Tables A and B are referred to as “group 1”compounds.

Compounds named in Table B are named by numbers assigned to R¹, R², R³and R⁴ according to the following compound naming convention,R¹.R².R³.R⁴, based on the numbered chemical substituents depicted inTable A. Each Table A number specifies a different structure for each ofR¹, R², R³ and R⁴. When R¹, R², R³ or R⁴ is a divalent moiety, e.g., ═O,the hydrogen at the corresponding position is absent. Thus, the group 1compound named 1.2.1.1 is a formula B structure with a β-hydroxyl bondedto carbons at the 3- and 7-positions (the variable groups R¹ and R²respectively), an α-bromine bonded to carbon 16 (the variable group R³)and double bonded oxygen (═O) at carbon 17 (the variable group R⁴),i.e., 1.2.1.1 has the structure shown below.

TABLE A R¹ R² 1 —OH 1 —H 2 ═O 2 —OH 3 —SH 3 ═O 4 ═S 4 —CH₃ 5 —O—CH₃ 5—OCH₃ 6 —O—S(O)(O)—O⁻Na⁺ 6 —OC₂H₅ 7 —O—S(O)(O)—OC₂H₅ 7 —OCH₂CH₂CH₃ 8—CH₃ 8 —OCH₂CH₂CH₂CH₃ 9 —H 9 —Cl 10 —OC(O)C(CH₃)₃ 10 —Br R³ R⁴ 1 —Br 1═O 2 —Cl 2 —OH 3 —I 3 —H 4 —F 4 —F 5 —H 5 —Cl 6 —OH 6 —Br 7 ═O 7 —I 8—O—C(O)—CH₃ 8 —O—C(O)—CH₃ 9 —O—C(O)—CH₂CH₃ 9 —O—C(O)—CH₂CH₃ 10—O—C(O)—CH₂CH₂CH₃ 10 —O—C(O)—CH₂CH₂CH₃

TABLE B 1.1.1.1, 1.1.1.2, 1.1.1.3, 1.1.1.4, 1.1.1.5, 1.1.1.6, 1.1.1.7,1.1.1.8, 1.1.1.9, 1.1.1.10, 1.1.2.1, 1.1.2.2, 1.1.2.3, 1.1.2.4, 1.1.2.5,1.1.2.6, 1.1.2.7, 1.1.2.8, 1.1.2.9, 1.1.2.10, 1.1.3.1, 1.1.3.2, 1.1.3.3,1.1.3.4, 1.1.3.5, 1.1.3.6, 1.1.3.7, 1.1.3.8, 1.1.3.9, 1.1.3.10, 1.1.4.1,1.1.4.2, 1.1.4.3, 1.1.4.4, 1.1.4.5, 1.1.4.6, 1.1.4.7, 1.1.4.8, 1.1.4.9,1.1.4.10, 1.1.5.1, 1.1.5.2, 1.1.5.3, 1.1.5.4, 1.1.5.5, 1.1.5.6, 1.1.5.7,1.1.5.8, 1.1.5.9, 1.1.5.10, 1.1.6.1, 1.1.6.2, 1.1.6.3, 1.1.6.4, 1.1.6.5,1.1.6.6, 1.1.6.7, 1.1.6.8, 1.1.6.9, 1.1.6.10, 1.1.7.1, 1.1.7.2, 1.1.7.3,1.1.7.4, 1.1.7.5, 1.1.7.6, 1.1.7.7, 1.1.7.8, 1.1.7.9, 1.1.7.10, 1.1.8.1,1.1.8.2, 1.1.8.3, 1.1.8.4, 1.1.8.5, 1.1.8.6, 1.1.8.7, 1.1.8.8, 1.1.8.9,1.1.8.10, 1.1.9.1, 1.1.9.2, 1.1.9.3, 1.1.9.4, 1.1.9.5, 1.1.9.6, 1.1.9.7,1.1.9.8, 1.1.9.9, 1.1.9.10, 1.1.10.1, 1.1.10.2, 1.1.10.3, 1.1.10.4,1.1.10.5, 1.1.10.6, 1.1.10.7, 1.1.10.8, 1.1.10.9, 1.1.10.10, 1.2.1.1,1.2.1.2, 1.2.1.3, 1.2.1.4, 1.2.1.5, 1.2.1.6, 1.2.1.7, 1.2.1.8, 1.2.1.9,1.2.1.10, 1.2.2.1, 1.2.2.2, 1.2.2.3, 1.2.2.4, 1.2.2.5, 1.2.2.6, 1.2.2.7,1.2.2.8, 1.2.2.9, 1.2.2.10, 1.2.3.1, 1.2.3.2, 1.2.3.3, 1.2.3.4, 1.2.3.5,1.2.3.6, 1.2.3.7, 1.2.3.8, 1.2.3.9, 1.2.3.10, 1.2.4.1, 1.2.4.2, 1.2.4.3,1.2.4.4, 1.2.4.5, 1.2.4.6, 1.2.4.7, 1.2.4.8, 1.2.4.9, 1.2.4.10, 1.2.5.1,1.2.5.2, 1.2.5.3, 1.2.5.4, 1.2.5.5, 1.2.5.6, 1.2.5.7, 1.2.5.8, 1.2.5.9,1.2.5.10, 1.2.6.1, 1.2.6.2, 1.2.6.3, 1.2.6.4, 1.2.6.5, 1.2.6.6, 1.2.6.7,1.2.6.8, 1.2.6.9, 1.2.6.10, 1.2.7.1, 1.2.7.2, 1.2.7.3, 1.2.7.4, 1.2.7.5,1.2.7.6, 1.2.7.7, 1.2.7.8, 1.2.7.9, 1.2.7.10, 1.2.8.1, 1.2.8.2, 1.2.8.3,1.2.8.4, 1.2.8.5, 1.2.8.6, 1.2.8.7, 1.2.8.8, 1.2.8.9, 1.2.8.10, 1.2.9.1,1.2.9.2, 1.2.9.3, 1.2.9.4, 1.2.9.5, 1.2.9.6, 1.2.9.7, 1.2.9.8, 1.2.9.9,1.2.9.10, 1.2.10.1, 1.2.10.2, 1.2.10.3, 1.2.10.4, 1.2.10.5, 1.2.10.6,1.2.10.7, 1.2.10.8, 1.2.10.9, 1.2.10.10, 1.3.1.1, 1.3.1.2, 1.3.1.3,1.3.1.4, 1.3.1.5, 1.3.1.6, 1.3.1.7, 1.3.1.8, 1.3.1.9, 1.3.1.10, 1.3.2.1,1.3.2.2, 1.3.2.3, 1.3.2.4, 1.3.2.5, 1.3.2.6, 1.3.2.7, 1.3.2.8, 1.3.2.9,1.3.2.10, 1.3.3.1, 1.3.3.2, 1.3.3.3, 1.3.3.4, 1.3.3.5, 1.3.3.6, 1.3.3.7,1.3.3.8, 1.3.3.9, 1.3.3.10, 1.3.4.1, 1.3.4.2, 1.3.4.3, 1.3.4.4, 1.3.4.5,1.3.4.6, 1.3.4.7, 1.3.4.8, 1.3.4.9, 1.3.4.10, 1.3.5.1, 1.3.5.2, 1.3.5.3,1.3.5.4, 1.3.5.5, 1.3.5.6, 1.3.5.7, 1.3.5.8, 1.3.5.9, 1.3.5.10, 1.3.6.1,1.3.6.2, 1.3.6.3, 1.3.6.4, 1.3.6.5, 1.3.6.6, 1.3.6.7, 1.3.6.8, 1.3.6.9,1.3.6.10, 1.3.7.1, 1.3.7.2, 1.3.7.3, 1.3.7.4, 1.3.7.5, 1.3.7.6, 1.3.7.7,1.3.7.8, 1.3.7.9, 1.3.7.10, 1.3.8.1, 1.3.8.2, 1.3.8.3, 1.3.8.4, 1.3.8.5,1.3.8.6, 1.3.8.7, 1.3.8.8, 1.3.8.9, 1.3.8.10, 1.3.9.1, 1.3.9.2, 1.3.9.3,1.3.9.4, 1.3.9.5, 1.3.9.6, 1.3.9.7, 1.3.9.8, 1.3.9.9, 1.3.9.10,1.3.10.1, 1.3.10.2, 1.3.10.3, 1.3.10.4, 1.3.10.5, 1.3.10.6, 1.3.10.7,1.3.10.8, 1.3.10.9, 1.3.10.10, 1.4.1.1, 1.4.1.2, 1.4.1.3, 1.4.1.4,1.4.1.5, 1.4.1.6, 1.4.1.7, 1.4.1.8, 1.4.1.9, 1.4.1.10, 1.4.2.1, 1.4.2.2,1.4.2.3, 1.4.2.4, 1.4.2.5, 1.4.2.6, 1.4.2.7, 1.4.2.8, 1.4.2.9, 1.4.2.10,1.4.3.1, 1.4.3.2, 1.4.3.3, 1.4.3.4, 1.4.3.5, 1.4.3.6, 1.4.3.7, 1.4.3.8,1.4.3.9, 1.4.3.10, 1.4.4.1, 1.4.4.2, 1.4.4.3, 1.4.4.4, 1.4.4.5, 1.4.4.6,1.4.4.7, 1.4.4.8, 1.4.4.9, 1.4.4.10, 1.4.5.1, 1.4.5.2, 1.4.5.3, 1.4.5.4,1.4.5.5, 1.4.5.6, 1.4.5.7, 1.4.5.8, 1.4.5.9, 1.4.5.10, 1.4.6.1, 1.4.6.2,1.4.6.3, 1.4.6.4, 1.4.6.5, 1.4.6.6, 1.4.6.7, 1.4.6.8, 1.4.6.9, 1.4.6.10,1.4.7.1, 1.4.7.2, 1.4.7.3, 1.4.7.4, 1.4.7.5, 1.4.7.6, 1.4.7.7, 1.4.7.8,1.4.7.9, 1.4.7.10, 1.4.8.1, 1.4.8.2, 1.4.8.3, 1.4.8.4, 1.4.8.5, 1.4.8.6,1.4.8.7, 1.4.8.8, 1.4.8.9, 1.4.8.10, 1.4.9.1, 1.4.9.2, 1.4.9.3, 1.4.9.4,1.4.9.5, 1.4.9.6, 1.4.9.7, 1.4.9.8, 1.4.9.9, 1.4.9.10, 1.4.10.1,1.4.10.2, 1.4.10.3, 1.4.10.4, 1.4.10.5, 1.4.10.6, 1.4.10.7, 1.4.10.8,1.4.10.9, 1.4.10.10, 1.5.1.1, 1.5.1.2, 1.5.1.3, 1.5.1.4, 1.5.1.5,1.5.1.6, 1.5.1.7, 1.5.1.8, 1.5.1.9, 1.5.1.10, 1.5.2.1, 1.5.2.2, 1.5.2.3,1.5.2.4, 1.5.2.5, 1.5.2.6, 1.5.2.7, 1.5.2.8, 1.5.2.9, 1.5.2.10, 1.5.3.1,1.5.3.2, 1.5.3.3, 1.5.3.4, 1.5.3.5, 1.5.3.6, 1.5.3.7, 1.5.3.8, 1.5.3.9,1.5.3.10, 1.5.4.1, 1.5.4.2, 1.5.4.3, 1.5.4.4, 1.5.4.5, 1.5.4.6, 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10.5.1.4, 10.5.1.5,10.5.1.6, 10.5.1.7, 10.5.1.8, 10.5.1.9, 10.5.1.10, 10.5.2.1, 10.5.2.2,10.5.2.3, 10.5.2.4, 10.5.2.5, 10.5.2.6, 10.5.2.7, 10.5.2.8, 10.5.2.9,10.5.2.10, 10.5.3.1, 10.5.3.2, 10.5.3.3, 10.5.3.4, 10.5.3.5, 10.5.3.6,10.5.3.7, 10.5.3.8, 10.5.3.9, 10.5.3.10, 10.5.4.1, 10.5.4.2, 10.5.4.3,10.5.4.4, 10.5.4.5, 10.5.4.6, 10.5.4.7, 10.5.4.8, 10.5.4.9, 10.5.4.10,10.5.5.1, 10.5.5.2, 10.5.5.3, 10.5.5.4, 10.5.5.5, 10.5.5.6, 10.5.5.7,10.5.5.8, 10.5.5.9, 10.5.5.10, 10.5.6.1, 10.5.6.2, 10.5.6.3, 10.5.6.4,10.5.6.5, 10.5.6.6, 10.5.6.7, 10.5.6.8, 10.5.6.9, 10.5.6.10, 10.5.7.1,10.5.7.2, 10.5.7.3, 10.5.7.4, 10.5.7.5, 10.5.7.6, 10.5.7.7, 10.5.7.8,10.5.7.9, 10.5.7.10, 10.5.8.1, 10.5.8.2, 10.5.8.3, 10.5.8.4, 10.5.8.5,10.5.8.6, 10.5.8.7, 10.5.8.8, 10.5.8.9, 10.5.8.10, 10.5.9.1, 10.5.9.2,10.5.9.3, 10.5.9.4, 10.5.9.5, 10.5.9.6, 10.5.9.7, 10.5.9.8, 10.5.9.9,10.5.9.10, 10.5.10.1, 10.5.10.2, 10.5.10.3, 10.5.10.4, 10.5.10.5,10.5.10.6, 10.5.10.7, 10.5.10.8, 10.5.10.9, 10.5.10.10, 10.6.1.1,10.6.1.2, 10.6.1.3, 10.6.1.4, 10.6.1.5, 10.6.1.6, 10.6.1.7, 10.6.1.8,10.6.1.9, 10.6.1.10, 10.6.2.1, 10.6.2.2, 10.6.2.3, 10.6.2.4, 10.6.2.5,10.6.2.6, 10.6.2.7, 10.6.2.8, 10.6.2.9, 10.6.2.10, 10.6.3.1, 10.6.3.2,10.6.3.3, 10.6.3.4, 10.6.3.5, 10.6.3.6, 10.6.3.7, 10.6.3.8, 10.6.3.9,10.6.3.10, 10.6.4.1, 10.6.4.2, 10.6.4.3, 10.6.4.4, 10.6.4.5, 10.6.4.6,10.6.4.7, 10.6.4.8, 10.6.4.9, 10.6.4.10, 10.6.5.1, 10.6.5.2, 10.6.5.3,10.6.5.4, 10.6.5.5, 10.6.5.6, 10.6.5.7, 10.6.5.8, 10.6.5.9, 10.6.5.10,10.6.6.1, 10.6.6.2, 10.6.6.3, 10.6.6.4, 10.6.6.5, 10.6.6.6, 10.6.6.7,10.6.6.8, 10.6.6.9, 10.6.6.10, 10.6.7.1, 10.6.7.2, 10.6.7.3, 10.6.7.4,10.6.7.5, 10.6.7.6, 10.6.7.7, 10.6.7.8, 10.6.7.9, 10.6.7.10, 10.6.8.1,10.6.8.2, 10.6.8.3, 10.6.8.4, 10.6.8.5, 10.6.8.6, 10.6.8.7, 10.6.8.8,10.6.8.9, 10.6.8.10, 10.6.9.1, 10.6.9.2, 10.6.9.3, 10.6.9.4, 10.6.9.5,10.6.9.6, 10.6.9.7, 10.6.9.8, 10.6.9.9, 10.6.9.10, 10.6.10.1, 10.6.10.2,10.6.10.3, 10.6.10.4, 10.6.10.5, 10.6.10.6, 10.6.10.7, 10.6.10.8,10.6.10.9, 10.6.10.10, 10.7.1.1, 10.7.1.2, 10.7.1.3, 10.7.1.4, 10.7.1.5,10.7.1.6, 10.7.1.7, 10.7.1.8, 10.7.1.9, 10.7.1.10, 10.7.2.1, 10.7.2.2,10.7.2.3, 10.7.2.4, 10.7.2.5, 10.7.2.6, 10.7.2.7, 10.7.2.8, 10.7.2.9,10.7.2.10, 10.7.3.1, 10.7.3.2, 10.7.3.3, 10.7.3.4, 10.7.3.5, 10.7.3.6,10.7.3.7, 10.7.3.8, 10.7.3.9, 10.7.3.10, 10.7.4.1, 10.7.4.2, 10.7.4.3,10.7.4.4, 10.7.4.5, 10.7.4.6, 10.7.4.7, 10.7.4.8, 10.7.4.9, 10.7.4.10,10.7.5.1, 10.7.5.2, 10.7.5.3, 10.7.5.4, 10.7.5.5, 10.7.5.6, 10.7.5.7,10.7.5.8, 10.7.5.9, 10.7.5.10, 10.7.6.1, 10.7.6.2, 10.7.6.3, 10.7.6.4,10.7.6.5, 10.7.6.6, 10.7.6.7, 10.7.6.8, 10.7.6.9, 10.7.6.10, 10.7.7.1,10.7.7.2, 10.7.7.3, 10.7.7.4, 10.7.7.5, 10.7.7.6, 10.7.7.7, 10.7.7.8,10.7.7.9, 10.7.7.10, 10.7.8.1, 10.7.8.2, 10.7.8.3, 10.7.8.4, 10.7.8.5,10.7.8.6, 10.7.8.7, 10.7.8.8, 10.7.8.9, 10.7.8.10, 10.7.9.1, 10.7.9.2,10.7.9.3, 10.7.9.4, 10.7.9.5, 10.7.9.6, 10.7.9.7, 10.7.9.8, 10.7.9.9,10.7.9.10, 10.7.10.1, 10.7.10.2, 10.7.10.3, 10.7.10.4, 10.7.10.5,10.7.10.6, 10.7.10.7, 10.7.10.8, 10.7.10.9, 10.7.10.10, 10.8.1.1,10.8.1.2, 10.8.1.3, 10.8.1.4, 10.8.1.5, 10.8.1.6, 10.8.1.7, 10.8.1.8,10.8.1.9, 10.8.1.10, 10.8.2.1, 10.8.2.2, 10.8.2.3, 10.8.2.4, 10.8.2.5,10.8.2.6, 10.8.2.7, 10.8.2.8, 10.8.2.9, 10.8.2.10, 10.8.3.1, 10.8.3.2,10.8.3.3, 10.8.3.4, 10.8.3.5, 10.8.3.6, 10.8.3.7, 10.8.3.8, 10.8.3.9,10.8.3.10, 10.8.4.1, 10.8.4.2, 10.8.4.3, 10.8.4.4, 10.8.4.5, 10.8.4.6,10.8.4.7, 10.8.4.8, 10.8.4.9, 10.8.4.10, 10.8.5.1, 10.8.5.2, 10.8.5.3,10.8.5.4, 10.8.5.5, 10.8.5.6, 10.8.5.7, 10.8.5.8, 10.8.5.9, 10.8.5.10,10.8.6.1, 10.8.6.2, 10.8.6.3, 10.8.6.4, 10.8.6.5, 10.8.6.6, 10.8.6.7,10.8.6.8, 10.8.6.9, 10.8.6.10, 10.8.7.1, 10.8.7.2, 10.8.7.3, 10.8.7.4,10.8.7.5, 10.8.7.6, 10.8.7.7, 10.8.7.8, 10.8.7.9, 10.8.7.10, 10.8.8.1,10.8.8.2, 10.8.8.3, 10.8.8.4, 10.8.8.5, 10.8.8.6, 10.8.8.7, 10.8.8.8,10.8.8.9, 10.8.8.10, 10.8.9.1, 10.8.9.2, 10.8.9.3, 10.8.9.4, 10.8.9.5,10.8.9.6, 10.8.9.7, 10.8.9.8, 10.8.9.9, 10.8.9.10, 10.8.10.1, 10.8.10.2,10.8.10.3, 10.8.10.4, 10.8.10.5, 10.8.10.6, 10.8.10.7, 10.8.10.8,10.8.10.9, 10.8.10.10, 10.9.1.1, 10.9.1.2, 10.9.1.3, 10.9.1.4, 10.9.1.5,10.9.1.6, 10.9.1.7, 10.9.1.8, 10.9.1.9, 10.9.1.10, 10.9.2.1, 10.9.2.2,10.9.2.3, 10.9.2.4, 10.9.2.5, 10.9.2.6, 10.9.2.7, 10.9.2.8, 10.9.2.9,10.9.2.10, 10.9.3.1, 10.9.3.2, 10.9.3.3, 10.9.3.4, 10.9.3.5, 10.9.3.6,10.9.3.7, 10.9.3.8, 10.9.3.9, 10.9.3.10, 10.9.4.1, 10.9.4.2, 10.9.4.3,10.9.4.4, 10.9.4.5, 10.9.4.6, 10.9.4.7, 10.9.4.8, 10.9.4.9, 10.9.4.10,10.9.5.1, 10.9.5.2, 10.9.5.3, 10.9.5.4, 10.9.5.5, 10.9.5.6, 10.9.5.7,10.9.5.8, 10.9.5.9, 10.9.5.10, 10.9.6.1, 10.9.6.2, 10.9.6.3, 10.9.6.4,10.9.6.5, 10.9.6.6, 10.9.6.7, 10.9.6.8, 10.9.6.9, 10.9.6.10, 10.9.7.1,10.9.7.2, 10.9.7.3, 10.9.7.4, 10.9.7.5, 10.9.7.6, 10.9.7.7, 10.9.7.8,10.9.7.9, 10.9.7.10, 10.9.8.1, 10.9.8.2, 10.9.8.3, 10.9.8.4, 10.9.8.5,10.9.8.6, 10.9.8.7, 10.9.8.8, 10.9.8.9, 10.9.8.10, 10.9.9.1, 10.9.9.2,10.9.9.3, 10.9.9.4, 10.9.9.5, 10.9.9.6, 10.9.9.7, 10.9.9.8, 10.9.9.9,10.9.9.10, 10.9.10.1, 10.9.10.2, 10.9.10.3, 10.9.10.4, 10.9.10.5,10.9.10.6, 10.9.10.7, 10.9.10.8, 10.9.10.9, 10.9.10.10, 10.10.1.1,10.10.1.2, 10.10.1.3, 10.10.1.4, 10.10.1.5, 10.10.1.6, 10.10.1.7,10.10.1.8, 10.10.1.9, 10.10.1.10, 10.10.2.1, 10.10.2.2, 10.10.2.3,10.10.2.4, 10.10.2.5, 10.10.2.6, 10.10.2.7, 10.10.2.8, 10.10.2.9,10.10.2.10, 10.10.3.1, 10.10.3.2, 10.10.3.3, 10.10.3.4, 10.10.3.5,10.10.3.6, 10.10.3.7, 10.10.3.8, 10.10.3.9, 10.10.3.10, 10.10.4.1,10.10.4.2, 10.10.4.3, 10.10.4.4, 10.10.4.5, 10.10.4.6, 10.10.4.7,10.10.4.8, 10.10.4.9, 10.10.4.10, 10.10.5.1, 10.10.5.2, 10.10.5.3,10.10.5.4, 10.10.5.5, 10.10.5.6, 10.10.5.7, 10.10.5.8, 10.10.5.9,10.10.5.10, 10.10.6.1, 10.10.6.2, 10.10.6.3, 10.10.6.4, 10.10.6.5,10.10.6.6, 10.10.6.7, 10.10.6.8, 10.10.6.9, 10.10.6.10, 10.10.7.1,10.10.7.2, 10.10.7.3, 10.10.7.4, 10.10.7.5, 10.10.7.6, 10.10.7.7,10.10.7.8, 10.10.7.9, 10.10.7.10, 10.10.8.1, 10.10.8.2, 10.10.8.3,10.10.8.4, 10.10.8.5, 10.10.8.6, 10.10.8.7, 10.10.8.8, 10.10.8.9,10.10.8.10, 10.10.9.1, 10.10.9.2, 10.10.9.3, 10.10.9.4, 10.10.9.5,10.10.9.6, 10.10.9.7, 10.10.9.8, 10.10.9.9, 10.10.9.10, 10.10.10.1,10.10.10.2, 10.10.10.3, 10.10.10.4, 10.10.10.5, 10.10.10.6, 10.10.10.7,10.10.10.8, 10.10.10.9, 10.10.10.10

Additional exemplary formula B compound groups include the followingcompound groups disclosed below. Unless otherwise specified, theconfigurations of all hydrogen atoms and R groups for the followingcompound groups are as defined for the group 1 compounds of formula Babove.

Group 2. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus described for group 1compounds, except that a double bond at the 5-6 position is present.Thus, group 2 compound 1.3.1.1 has the structure

Group 3. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus as described for group 1compounds, except that double bonds at the 1-2- and 5-6 positions arepresent. Thus, group 3 compound 2.2.5.1 has the structure

Group 4. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus described for group 1compounds, except that a double bond at the 1-2 position is present.Thus, group 4 compound 5.2.7.8 has the structure

Group 5. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus described for group 1compounds, except that a double bond at the 4-5 position is present.Thus, the group 5 compound named 3.5.2.9 has the structure

Group 6. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus described for group 1compounds, except that double bonds at both the 1-2 and 4-5 positionsare present. Thus, the group 6 compound named 10.2.7.8 has the structure

Group 7. Group 7 comprises the 6 compound groups described above, exceptthat R⁵ is hydrogen instead of methyl, i.e., it comprises 6 subgroups,7-1, 7-2, 7-3, 7-4, 7-5 and 7-6. Thus, subgroup 7-1 has the same steroidnucleus as group 1 above, i.e., no double bond is present, but R⁵ is —H.Subgroup 7-2 comprises the same steroid nucleus as group 2 above, i.e.,a double bond is present at the 5-6-position, but R⁵ is —H, Compoundsubgroups 7-3 through 7-6 are assigned a steroid nucleus in the samemanner. Thus, the subgroup 7-1 through subgroup 7-6 compounds named1.2.1.9 have the structures

Group 8. Group 8 comprises 6 subgroups of compounds, i.e., each compoundnamed in groups 1-6, except that R⁵ of formula B is —CH₂OH instead ofmethyl. The subgroups 8-1 through subgroup 8-6 compounds have structuresthat are named in the same manner as group 1-6 compounds, except that—CH₂OH instead of methyl is present at R⁵. These groups are named inessentially the same manner as subgroups 7-1 through 7-6. Thus, subgroup8-1 and subgroup 8-2 compounds named 1.2.1.9 have the structures

Group 9. Group 9 comprises each compound named in compound groups 1-8,except that R⁶ of formula B is hydrogen instead of methyl. Thus group 9comprises subgroups 9-1 through 9-8-6, i.e., 9-1, 9-2, 9-3, 9-4, 9-5,9-6, 9-7-1, 9-7-2, 9-7-3, 9-7-4, 9-7-5, 9-7-6, 9-8-1, 9-8-2, 9-8-3,9-8-4, 9-8-5 and 9-8-6. Subgroups 9-1 through 9-8-6 compounds havestructures that are named in essentially the same manner as subgroup 7-1through 7-6 compounds, except that —H instead of methyl is present atR⁶. Thus, subgroup 9-1 and subgroup 9-2 compounds named 1.2.1.9 have thestructures

Subgroup 9-7-1 compound 1.2.1.9 has the same structure as group 9-1compound 1.2.1.9, except that R⁵ is hydrogen in the β configuration,instead of a methyl group in the β configuration. Similarly, the group9-8-1 compound 1.2.1.9 has the same structure as group 9-1 compound1.2.1.9, except that R⁵ is hydroxymethyl (—CH₂OH) in the βconfiguration, instead of a methyl group in the β configuration. Group9-7-2 compound 1.2.1.9 has the same structure as the group 9-7-1compound, except that a double bond is present at the 5-6 position.

Thus, subgroups 9-1 through 9-6 have hydrogen at R⁶, but each has adifferent double bond structure, e.g., no double bond in subgroup 9-1and double bonds at 1-2 and 4-5 in subgroup 9-6. Subgroups 9-7-1 through9-7-6 also comprises six subgroups, but they have hydrogen at R⁵ and R⁶,but each has a different double bond structure for each of the sixsubgroups, e.g., no double bond in subgroup 9-7-1 and double bonds atpositions 1-2 and 4-5 in subgroup 9-7-6. Subgroups 9-8-1 through 9-8-6all have hydrogen at R⁶ and —CH₂OH at R⁵, but each has a differentdouble bond structure in each, e.g., no double bond in subgroup 9-8-1and double bonds at positions 1-2 and 4-5 in group 9-8-6.

Groups 10. Group 10 comprises each compound named in groups 1 through 8,but where R⁶ of formula B is —CH₂OH instead of methyl. The subgroups10-1 through group 10-6 compounds have structures that are named inessentially the same manner as compounds in group 9, except that —CH₂OHinstead of methyl is present at R⁶. Thus, subgroup 10-1 and subgroup10-2 compounds named 1.2.1.9 have the structures

Subgroup 10-7-1 compound 1.2.1.9 has the same structure as subgroup 10-1compound 1.2.1.9, except that R⁵ is hydrogen in the β configuration,instead of a methyl group in the β configuration. Similarly, thesubgroup 10-8-1 compound 1.2.1.9 has the same structure as group 10-1compound 1.2.1.9, except that R⁵ is hydroxymethyl (—CH₂OH) in the βconfiguration, instead of a methyl group in the β configuration.Subgroup 10-7-2 compound 1.2.1.9 has the same structure as the subgroup10-7-1 compound, except that a double bond is present at the 5-6position.

Thus, subgroups 10-1 through 10-8-6 comprise 18 separate groups, each ofwhich has —CH₂OH at R⁶. Subgroups 10-1 through 10-6 comprise differentsix subgroups where each has a different double bond structure, e.g., nodouble bond in subgroup 10-1 and double bonds at 1-2 and 4-5 in subgroup10-6. Subgroups 10-7-1 through 10-7-6 all have —CH₂OH at R⁶ and hydrogenat R⁵, but each has a different double bond structure for each of thesix groups, e.g., no double bond in subgroup 10-7-1 and double bonds atpositions 1-2 and 4-5 in subgroup 10-7-6. Similarly, subgroups 10-8-1through 10-8-6 all six have —CH₂OH at R⁶ and at R⁵, but each has adifferent double bond structure in each of the six subgroups, e.g., nodouble bond in subgroup 10-8-1 and double bonds at positions 1-2 and 4-5in subgroup 10-8-6. The 18 groups are 10-1, 10-2, 10-3, 10-4, 10-5,10-6, 10-7-1, 10-7-2, 10-7-3, 10-7-4, 10-7-5, 10-7-6, 10-8-1, 10-8-2,10-8-3, 10-8-4, 10-8-5 and 10-8-6.

Group 11. Group 11 comprises each compound named in compound groups1-10, but where R¹ moieties (or substituents) 1-10 listed in Table A arereplaced with the following moieties:

-   1-O—C(O)—CH₂CH₂CH₂CH₃ (—O—C(O)—CH₂CH₂CH₂CH₃ replaces —OH, which is    R¹ moiety 1 in Table A)-   2 —O—C(O)—CH₂CH₂CH₂CH₂CH₂CH₃-   3 —O—C(O)—CH₂CH₂OCH₂CH₃-   4 —O—C(O)—CH₂CH₂OCH₂CH₂OCH₂CH₃-   5 —O—C(O)—CH₂CH₂CH₂CH₂OCH₂CH₃-   6 —O—C(O)—CH₂CH₂OCH₂CH₂CH₂CH₃-   7 —O—C₆H₄C₁₋₈-   8 —O—C₆H₃F₂-   9 —O—C₆H₄—O(CH₂)₂—O—CH₂CH₃-   10 —O—C₆H₄—C(O)O(CH₂)₀₋₉CH₃

The subgroup 11-1 through subgroup 11-6 compounds have structures thatare named in essentially the same manner as described for the groupsabove, except that moieties 1-10 of table A are replaced by the moieties1-10 at R¹. Thus subgroup 11-1 and 11-2 compounds named 1.2.1.9 have thestructures

Subgroup 11-7-1 and 11-7-2 compounds named 1.2.1.9 have the structures

Subgroup 11-8-1 and 11-8-2 compounds named 1.2.1.9 have the structures

Group 11 comprises 54 separate subgroups, subgroups 11-1 through11-10-8-6, where each of which has the R¹ moieties shown in this groupand the remaining moieties as shown in the other groups described above.Subgroups 11-1 through 11-6 each have a different double bond structure,e.g., no double bond in subgroup 11-1 and double bonds at 1-2 and 4-5 insubgroup 11-6. Subgroups 11-7-1 through 11-7-6 all have —CH₂OH at R⁶ andhydrogen at R⁵, but each has a different double bond structure, e.g., nodouble bond in subgroup 11-7-1 and double bonds at positions 1-2 and 4-5in subgroup 11-7-6. Subgroups 11-8-1 through 11-8-6 comprise all have—CH₂OH at R⁶ and at R⁵, but each has a different double bond structurein each of the six groups, e.g., no double bond in group 11-8-1 anddouble bonds at positions 1-2 and 4-5 in group 11-8-6. The compounds inthe remaining groups are named in essentially the same manner.

The 54 groups are 11-1, 11-2, 11-3, 11-4, 11-5, 11-6, 11-7-1, 11-7-2,11-7-3, 11-7-4, 11-7-5, 11-7-6, 11-8-1, 11-8-2, 11-8-3, 11-8-4, 11-8-5,11-8-6, 11-9-1, 11-9-2, 11-9-3, 11-9-4, 11-9-5, 11-9-6, 11-10-1,11-10-2, 11-10-3, 11-10-4, 11-10-5, 11-10-6, 11-9-7-1, 11-9-7-2,11-9-7-3, 11-9-7-4, 11-9-7-5, 11-9-7-6, 11-10-7-1, 11-10-7-2, 11-10-7-3,11-10-7-4, 11-10-7-5, 11-10-7-6, 11-9-8-1, 11-9-8-2, 11-9-8-3, 11-9-8-4,11-9-8-5, 11-9-8-6, 11-10-8-1, 11-10-8-2, 11-10-8-3, 11-10-8-4,11-10-8-5 and 11-10-8-6.

Group 12. Group 12 comprises each compound named in groups 1 through 10,but where R¹ moieties 1-10 listed in Table A are replaced with thefollowing moieties:

-   1-O—P(O)(O)—OCH₂CH(CH₃)CH₃ (—O—P(O)(O)—OCH₂CH(CH₃)CH₃ replaces —OH,    which is R¹ moiety 1 in Table A)-   2 —O—P(O)(O)—OCH₂CH₂CH₂CH₂CH₃-   3 —O—P(O)(O)—OCH₂CH₂CH₂CH₂CH₂CH₃-   4 —O—P(O)(O)—OCH₂CH₂CH(CH₂CH₂)CH₃-   5 —O—CH₂CH₂CH₂CH₂CH₂CH₃-   6 —O—C1-C6 alkyl(OH)₀₋₂-   7 —C1-C6 alkyl(OH)₀₋₂-   8 —C(O)—C1-C6 alkyl(OH)₀₋₂-   9 —O-monosaccharide-   10 —O-disaccharide

Group 12 comprises 54 separate subgroups, subgroups 12-1 through12-10-8-6, where each of which has the R¹ moieties shown in this groupand the remaining moieties as shown in the other groups described above.The subgroups are defined essentially as described for group 11 above.The 54 subgroups are 12-1, 12-2, 12-3, 12-4, 12-5, 12-6, 12-7-1, 12-7-2,12-7-3, 12-7-4, 12-7-5, 12-7-6, 12-8-1, 12-8-2, 12-8-3, 12-8-4, 12-8-5,12-8-6, 12-9-1, 12-9-2, 12-9-3, 12-9-4, 12-9-5, 12-9-6, 12-10-1,12-10-2, 12-10-3, 12-10-4, 12-10-5, 12-10-6, 12-9-7-1, 12-9-7-2,12-9-7-3, 12-9-7-4, 12-9-7-5, 12-9-7-6, 12-10-7-1, 12-10-7-2, 12-10-7-3,12-10-7-4, 12-10-7-5, 12-10-7-6, 12-9-8-1, 12-9-8-2, 12-9-8-3, 12-9-8-4,12-9-8-5, 12-9-8-6, 12-10-8-1, 12-10-8-2, 12-10-8-3, 12-10-8-4,12-10-8-5 and 12-10-8-6.

Group 13. Group 13 comprises each compound named in groups 1 through 10,but where R¹ moieties 1-10 listed in Table A are replaced with thefollowing moieties:

-   1 —O—(CH₂)₄—CH₃(—O—(CH₂)₄—CH₃ replaces —OH, which is R¹ moiety 1 in    Table A)-   2 —O-oligosaccharide-   3 —O-polyethylene glycol (e.g., PEG20, PEG100, PEG200 or PEG400)-   4 —O—C(O)—NH₀₋₂(C1-C6 alkyl)₀₋₂-   5 —C(O)—NH₀₋₂(C1-C6 alkyl)₀₋₂-   6 —O—C(O)—NH(CH₂)₂₋₄—O—C1-C4 alkyl(OH)₀₋₂-   7 —O—C(O)—CH₃-   8 —O—C(O)—C2-C5 alkyl(OH)₀₋₂-   9 —O—C(O)—CH₂CH₂CH₂CH₃-   10 —O—C(O)—CH(NH₂)—R⁴² (R⁴² is —H, C₂-C₆ alkyl or an amino acid side    chain)

Group 13 comprises 54 separate subgroups, subgroups 13-1 through13-10-8-6, where each of which has the R¹ moieties shown in this groupand the remaining moieties as shown in the other groups described above.The subgroups are defined essentially as described for group 11 above.The 54 subgroups are 13-1, 13-2, 13-3, 13-4, 13-5, 13-6, 13-7-1, 13-7-2,13-7-3, 13-7-4, 13-7-5, 13-7-6, 13-8-1, 13-8-2, 13-8-3, 13-8-4, 13-8-5,13-8-6, 13-9-1, 13-9-2, 13-9-3, 13-9-4, 13-9-5, 13-9-6, 13-10-1,13-10-2, 13-10-3, 13-10-4, 13-10-5, 13-10-6, 13-9-7-1, 13-9-7-2,13-9-7-3, 13-9-7-4, 13-9-7-5, 13-9-7-6, 13-10-7-1, 13-10-7-2, 13-10-7-3,13-10-7-4, 13-10-7-5, 13-10-7-6, 13-9-8-1, 13-9-8-2, 13-9-8-3, 13-9-8-4,13-9-8-5, 13-9-8-6, 13-10-8-1, 13-10-8-2, 13-10-8-3, 13-10-8-4,13-10-8-5 and 13-10-8-6.

Group 14. Group 14 comprises each compound named in groups 1 through 10,but where R¹ moieties 1-10 listed in Table A are replaced with thefollowing moieties:

-   1 —C(O)—CH₃-   2 —O—CH₂C₆H₅-   3 —C(S)—CH₃-   4 —O—C0-C6 alkyl-heterocycle-   5 —C0-C6 alkyl-heterocycle-   6 —O—CH₂C₆H₄F-   7 —O—CH₂C₆H₃(OCH₃)₂-   8 —C(O)—C2-C4 alkyl-O—C1-C3 alkyl-   9 —C(O)—C0-C4 alkyl-NH—(C1-C3 alkyl)₀₋₁-H-   10 —O—CH₂C₆H₄OCH₂CH₃

Group 14 comprises 54 separate subgroups, subgroups 14-1 through14-10-8-6, where each of which has the R¹ moieties shown in this groupand the remaining moieties as shown in the other groups described above.These subgroups are defined essentially as described for group 11 above.The 54 subgroups are 14-1, 14-2, 14-3, 14-4, 14-5, 14-6, 14-7-1, 14-7-2,14-7-3, 14-7-4, 14-7-5, 14-7-6, 14-8-1, 14-8-2, 14-8-3, 14-8-4, 14-8-5,14-8-6, 14-9-1, 14-9-2, 14-9-3, 14-9-4, 14-9-5, 14-9-6, 14-10-1,14-10-2, 14-10-3, 14-10-4, 14-10-5, 14-10-6, 14-9-7-1, 14-9-7-2,14-9-7-3, 14-9-7-4, 14-9-7-5, 14-9-7-6, 14-10-7-1, 14-10-7-2, 14-10-7-3,14-10-7-4, 14-10-7-5, 14-10-7-6, 14-9-8-1, 14-9-8-2, 14-9-8-3, 14-9-8-4,14-9-8-5, 14-9-8-6, 14-10-8-1, 14-10-8-2, 14-10-8-3, 14-10-8-4,14-10-8-5 and 14-10-8-6.

Group 15. Group 15 comprises each compound named in groups 1 through 10,but where R¹ moieties 1-10 listed in Table A are replaced with thefollowing groups:

-   1 —O—C(O)—CH₂CH₂NH₂ (—O—C(O)—CH₂CH₂NH₂ replaces —OH, which is R¹    moiety 1 in Table A)-   2 —O—C(O)—C1-C6 alkyl-NH₂-   3 —C(O)—C1-C6 alkyl-NH₂-   4 —O—C(O)—C1-C6 alkyl-(OH)₀₋₂-   5 —C(O)—C1-C6 alkyl-(OH)₀₋₂-   6 —O—C(O)—C1-C6 alkyl-(SH)₀₋₂-   7 —C(O)—C1-C6 alkyl-(SH)₀₋₂-   8 —O—C(O)—CH₂CH₂CH₂SH-   9 —S—C(O)—C1-C6 alkyl-(OH)₀₋₂-   10 —C(S)—C1-C6 alkyl-(OH)₀₋₂

Group 15 comprises 54 separate subgroups, subgroups 15-1 through15-10-8-6, where each of which has the R¹ moieties shown in this groupand the remaining moieties as shown in the other groups described above.These subgroups are defined essentially as described for group 11 above.The 54 subgroups are 15-1, 15-2, 15-3, 15-4, 15-5, 15-6, 15-7-1, 15-7-2,15-7-3, 15-7-4, 15-7-5, 15-7-6, 15-8-1, 15-8-2, 15-8-3, 15-8-4, 15-8-5,15-8-6, 15-9-1, 15-9-2, 15-9-3, 15-9-4, 15-9-5, 15-9-6, 15-10-1,15-10-2, 15-10-3, 15-10-4, 15-10-5, 15-10-6, 15-9-7-1, 15-9-7-2,15-9-7-3, 15-9-7-4, 15-9-7-5, 15-9-7-6, 15-10-7-1, 15-10-7-2, 15-10-7-3,15-10-7-4, 15-10-7-5, 15-10-7-6, 15-9-8-1, 15-9-8-2, 15-9-8-3, 15-9-8-4,15-9-8-5, 15-9-8-6, 15-10-8-1, 15-10-8-2, 15-10-8-3, 15-10-8-4,15-10-8-5 and 15-10-8-6.

Group 16. Groups 16 comprises each compound named in groups 1 through10, but where R¹ moieties 1-10 listed in Table A are replaced with thefollowing groups:

-   1 —O—C(O)-A4-NH₂, where A4-NH₂ is a 4 carbon alkyl group substituted    with —NH₂ (—O—C(O)-A4-NH₂ replaces —OH, which is R¹ moiety 1 in    Table A)-   2 —O—C(O)-A6-NH₂, where A6-NH₂ is a 6 carbon alkyl group substituted    with —NH₂-   3 —O—C(O)-A8-NH₂, where A8-NH₂ is a 8 carbon alkyl group substituted    with —NH₂-   4 —O—C(O)-A4-OH, where A4-OH is a 4 carbon alkyl group substituted    with —OH or —O—-   5 —O—C(O)-A6-OH, where A6-OH is a 6 carbon alkyl group substituted    with —OH or —O—-   6-O—C(O)-A8-OH, where A8-OH is a 8 carbon alkyl group substituted    with —OH or —O—-   7 —F-   8 —C₁₋₉-   —Br-   10 —I

Group 16 comprises 54 separate subgroups, subgroups 16-1 through16-10-8-6, where each of which has the R¹ moieties shown in this groupand the remaining moieties as shown in the other groups described above.These groups are defined essentially as described for group 11 above.The 54 subgroups are 16-1, 16-2, 16-3, 16-4, 16-5, 16-6, 16-7-1, 16-7-2,16-7-3, 16-7-4, 16-7-5, 16-7-6, 16-8-1, 16-8-2, 16-8-3, 16-8-4, 16-8-5,16-8-6, 16-9-1, 16-9-2, 16-9-3, 16-9-4, 16-9-5, 16-9-6, 16-10-1,16-10-2, 16-10-3, 16-10-4, 16-10-5, 16-10-6, 16-9-7-1, 16-9-7-2,16-9-7-3, 16-9-7-4, 16-9-7-5, 16-9-7-6, 16-10-7-1, 16-10-7-2, 16-10-7-3,16-10-7-4, 16-10-7-5, 16-10-7-6, 16-9-8-1, 16-9-8-2, 16-9-8-3, 16-9-8-4,16-9-8-5, 16-9-8-6, 16-10-8-1, 16-10-8-2, 16-10-8-3, 16-10-8-4,16-10-8-5 and 16-10-8-6.

Group 17. Group 17 comprises each compound named in compound groups 1through 10, but where R¹ moieties 1-10 listed in Table A are replacedwith the following groups:

-   1 —O—S(O)(O)—O—C1-C8 optionally substituted alkyl-   2 —O—P(O)(OH)—O—C1-C8 optionally substituted alkyl-   3 —O—P(S)(OH)—O—C1-C8 optionally substituted alkyl-   4 —O—P(O)(OH)—S—C1-C8 optionally substituted alkyl-   5 —O—S(O)(O)—OR⁴⁴ (R⁴⁴ is H, NH₄ ⁺, Na⁺, K⁺, HN⁺(CH₃)₄, N⁺(CH₃)₄,    HN⁺(C2H₅)₃ C1-C8 alkyl (e.g., —CH₃, —C2H₅ or —C3H₇), or pyridinium⁺)-   6 —O—P(O)(OH)—OR⁴⁴-   7 —O—P(O)(OH)—SR⁴⁴-   8 —O—S(O)(O)—O-2′,3′-dipalmitoyl-1′-glyceryl-   9 —O-(3β-O-1β)-D-glucuronic acid-R⁴⁴-   10 —O-(3β-O-1β)-tri-O-acetyl-D-glucuronic acid-R⁴⁴

Group 17 comprises 54 separate subgroups, subgroups 17-1 through17-10-8-6, where each of which has the R¹ moieties shown in this groupand the remaining moieties as shown in the other groups described above.These subgroups are defined essentially as described for group 11 above.The 54 subgroups are 17-1, 17-2, 17-3, 17-4, 17-5, 17-6, 17-7-1, 17-7-2,17-7-3, 17-7-4, 17-7-5, 17-7-6, 17-8-1, 17-8-2, 17-8-3, 17-8-4, 17-8-5,17-8-6, 17-9-1, 17-9-2, 17-9-3, 17-9-4, 17-9-5, 17-9-6, 17-10-1,17-10-2, 17-10-3, 17-10-4, 17-10-5, 17-10-6, 17-9-7-1, 17-9-7-2,17-9-7-3, 17-9-7-4, 17-9-7-5, 17-9-7-6, 17-10-7-1, 17-10-7-2, 17-10-7-3,17-10-7-4, 17-10-7-5, 17-10-7-6, 17-9-8-1, 17-9-8-2, 17-9-8-3, 17-9-8-4,17-9-8-5, 17-9-8-6, 17-10-8-1, 17-10-8-2, 17-10-8-3, 17-10-8-4,17-10-8-5 and 17-10-8-6.

Group 18. Group 18 comprises each compound named in groups 1 through 17,but where R⁴ moieties 1-10 listed in Table A are replaced with thefollowing moieties:

-   1 —O—C(O)CH₂NH₂-   2 —O—C(O)C(CH₃)H—NH₂-   3 —O—C(O)C(CH₂C₆H₅)H—NH₂-   4 —O—C(O)—O—NHC(CH₃)H—CO₂H-   5 —O—C(O)—O—NHCH₂—CO₂H-   6 —O—C(O)—O—NH(CH₂C₆H₅)H—CO₂H-   7 —O—C(O)—CF₃-   8 —O—C(O)—CH₂CF₃-   9 —O—C(O)—(CH₂)₃CF₃-   10 —O—C(O)—(CH₂)₅CH₃

Group 18 comprises 432 separate subgroups, 18-1 through 18-17-10-8-6,where each of which has the R⁴ moieties shown in this group and theremaining moieties as shown in the other groups described above. Thesegroups are defined essentially as described for the groups describedabove. The groups are 18-1 through 18-6, 18-7-1 through 18-7-6, 18-8-1through 18-8-6, 18-9-1 through 18-9-6, 18-10-1 through 18-10-6, 18-9-7-1through 18-9-7-6, 18-9-8-1 through 18-9-8-6, 18-10-7-1 through18-10-7-6, 18-10-8-1 through 18-10-8-6, 18-11-1 through 18-11-6,18-11-7-1 through 18-11-7-6, 18-11-8-1 through 18-11-8-6, 18-11-9-1through 18-11-9-6, 18-11-10-1 through 18-11-10-6, 18-11-9-7-1 through18-11-9-7-6, 18-11-9-8-1 through 18-11-9-8-6, 18-11-10-7-1 through18-11-10-7-6, 18-11-10-8-1 through 18-11-10-8-6, 18-12-1 through18-12-6, 18-12-7-1 through 18-12-7-6, 18-12-8-1 through 18-12-8-6,18-12-9-1 through 18-12-9-6, 18-12-10-1 through 18-12-10-6, 18-12-9-7-1through 18-12-9-7-6, 18-12-9-8-1 through 18-12-9-8-6, 18-12-10-7-1through 18-12-10-7-6, 18-12-10-8-1 through 18-12-10-8-6, 18-13-1 through18-13-6, 18-13-7-1 through 18-13-7-6, 18-13-8-1 through 18-13-8-6,18-13-9-1 through 18-13-9-6, 18-13-10-1 through 18-13-10-6, 18-13-9-7-1through 18-13-9-7-6, 18-13-9-8-1 through 18-13-9-8-6, 18-13-10-7-1through 18-13-10-7-6, 18-13-10-8-1 through 18-13-10-8-6, 18-14-1 through18-14-6, 18-14-7-1 through 18-14-7-6, 18-14-8-1 through 18-14-8-6,18-14-9-1 through 18-14-9-6, 18-14-10-1 through 18-14-10-6, 18-14-9-7-1through 18-14-9-7-6, 18-14-9-8-1 through 18-14-9-8-6, 18-14-10-7-1through 18-14-10-7-6, 18-14-10-8-1 through 18-14-10-8-6, 18-15-1 through18-15-6, 18-15-7-1 through 18-15-7-6, 18-15-8-1 through 18-15-8-6,18-15-9-1 through 18-15-9-6, 18-15-10-1 through 18-15-10-6, 18-15-9-7-1through 18-15-9-7-6, 18-15-9-8-1 through 18-15-9-8-6, 18-15-10-7-1through 18-15-10-7-6, 18-15-10-8-1 through 18-15-10-8-6, 18-16-1 through18-16-6, 18-16-7-1 through 18-16-7-6, 18-16-8-1 through 18-16-8-6,18-16-9-1 through 18-16-9-6, 18-16-10-1 through 18-16-10-6, 18-16-9-7-1through 18-16-9-7-6, 18-16-9-8-1 through 18-16-9-8-6, 18-16-10-7-1through 18-16-10-7-6, 18-16-10-8-1 through 18-16-10-8-6, 18-17-1 through18-17-6, 18-17-7-1 through 18-17-7-6, 18-17-8-1 through 18-17-8-6,18-17-9-1 through 18-17-9-6, 18-17-10-1 through 18-17-10-6, 18-17-9-7-1through 18-17-9-7-6, 18-17-9-8-1 through 18-17-9-8-6, 18-17-10-7-1through 18-17-10-7-6 and 18-17-10-8-1 through 18-17-10-8-6.

Group 19. Group 19 comprises each compound named in compound groups 1through 17, but where R⁴ moieties 1-10 listed in Table A are replacedwith the following moieties:

-   1 —O—C(O)—O—CH₃-   2 —O—C(O)—O—CH₂CH₃-   3 —O—C(O)—O—C₃H₇-   4 —O—C(O)—O—C₄H₉-   5 —O—C(O)—O—C₆H₁₃-   6 —O—C(O)—O—C₆H₅-   7 —O—C(O)—O—C₆H₄OH-   8 —O—C(O)—O—C₆H₄OCH₃-   9 —O—C(O)—O—C₆H₄OCH₂CH₃-   10 —O—C(O)—O—C₆H₄F

Group 19 comprises 432 separate subgroups, 19-1 through 19-17-10-8-6,where each of which has the R⁴ moieties shown in this group and theremaining moieties as shown in the other groups described above. Thesesubgroups are defined essentially as described for group 18 above. Thesubgroups are 19-1 through 19-6, 19-7-1 through 19-7-6, 19-8-1 through19-8-6, 19-9-1 through 19-9-6 and so on essentially as described forgroup 18 compounds.

Group 20. Group 20 comprises each compound named in groups 1 through 17,but where R⁴ moieties 1-10 listed in Table A are replaced with thefollowing moieties:

-   1 —O—S(O)(O)—OR⁴⁴ (R⁴⁴ is H, NH₄ ⁺, Na⁺, K⁺, HN⁺(CH₃)₃, N⁺(CH₃)₄,    HN⁺(C₂H₅)₃ C1-C8 optionally substituted alkyl (e.g., —CH₃, —C₂H₅ or    —C₃H₇), or pyridinium⁺)-   2 —O—P(O)(OH)—SR⁴⁴-   3 —C(O)—C₁-C₈ optionally substituted alkyl-   4 —CH(OH)—C₁-C₈ optionally substituted alkyl-   5 —C≡CH-   6 —C═C—(CH₂)₁₋₄—H-   7 —C(O)—CH₂—OH-   8 —C(S)—CH₂—OH-   9 —O—S(O)(O)—O-2′,3′-dipalmitoyl-1′-glyceryl-   10 —O-(3-O-1β)-tri-O-acetyl-D-glucuronic acid-R⁴⁴

Group 20 comprises 432 separate subgroups, 20-1 through 20-17-10-8-6comprise 432 separate groups, each of which has the R⁴ moieties definedfor this group and the remaining moieties as shown in the other groupsdescribed above. These subgroups are defined essentially as describedfor group 18 above. The subgroups are 20-1 through 20-6, 20-7-1 through20-7-6, 20-8-1 through 20-8-6, 20-9-1 through 20-9-6 and so onessentially as described for group 18 compounds.

Group 21. Group 21 comprises each compound named in compound groups 1through 17, but where R⁴ moieties 1-10 listed in Table A are replacedwith the following moieties:

-   1 —O—C(S)—O—C1-C4 alkyl-   2 —S—C(S)—O—C1-C4 alkyl-   3 —SH-   4 ═S-   5 —O—C1-C6 optionally substituted alkyl-   6 —O—C1-C6-optionally substituted alkyl-optionally substituted aryl-   7 —S—C1-C6 optionally substituted alkyl-   8 —O—C(O)—CH(NH₂)—R⁴² (R⁴² is —H, C2-C6 alkyl or an amino acid side    chain)-   9 —C0-C4 alkyl-heterocycle-   10 —O-polyethylene glycol (e.g., PEG20, PEG100, PEG200 or PEG400)

Group 21 comprises 432 separate subgroups, 21-1 through 21-17-10-8-6comprise 432 separate groups, each of which has the R⁴ moieties definedfor this group and the remaining moieties as shown in the other groupsdescribed above. These subgroups are defined essentially as describedfor group 18 above. The subgroups are 21-1 through 21-6, 21-7-1 through21-7-6, 21-8-1 through 21-8-6, 21-9-1 through 21-9-6 and so onessentially as described for group 18 compounds.

Group 22. Group 22 comprises each compound named in compound groups 1through 21, but where R² moieties 1-10 listed in Table A are replacedwith the following moieties:

-   1 —O—C(S)—O—C1-C8 alkyl-(OH)₀₋₂-   2 —O—C(O)—O—C1-C8 alkyl-(OH)₀₋₂-   3 —C(O)—C1-C6 alkyl-O—C1-C2 alkyl-   4 —C(O)—C1-C6 alkyl-(S)₀₋₁—C1-C2 alkyl-(OH)₀₋₁-   5 —C(O)—C1-C6 alkyl-NH₀₋₂(C1-C4 alkyl)₀₋₂-   6 —O—C(O)—C0-C4 alkyl-heterocycle-   7 —C(O)—O—C1-C4 alkyl-C6H₃₋₅—(OH)₀₋₂-   8 —O—C(O)—O—C1-C4 alkyl-C6H₃₋₅—(OH)₀₋₂-   9 —O—C(O)—C1-C4 alkyl-C6H₃₋₅—(O—C1-C4 alkyl)₀₋₂-   10 —O—C(O)—C1-C4 alkyl-C6H₃₋₅-(halogen)₀₋₂

Group 22 comprises subgroups 22-1 through 22-21-17-10-8-6, which namecompounds or genera of compounds essentially as described for the othercompound groups above. The 1728 subgroups in group 22 are 22-1 through22-6, 22-7-1 through 22-7-6, 22-8-1 through 22-8-6, 22-9-1 through22-9-6 and so on essentially as described for the groups above.

Group 23. Group 23 comprises each compound named in compound groups 1through 21, but where R² moieties 1-10 listed in Table A are replacedwith the following moieties:

-   1 —O—C₀₋₄ alkyl-heterocycle-   2 —O—C(O)—C₀₋₄ alkyl-heterocycle-   3 —SH-   4 ═S-   5 —C2-C6 alkyl-(OH)₁₋₂-   6 —O—CHR²⁴—C(O)—R²⁵-   7 —O—CHR²⁴—C(O)—N(R²⁵)₂-   8 —O—CHR²⁴—C(O)—NHR²⁵-   9 —O—CHR²⁴—C(O)—NH₂-   10 —O—CHR²⁴—C(O)—OC₆H₅

Group 23 comprises subgroups 24-1 through 24-23-21-17-10-8-6, which namecompounds or genera of compounds essentially as described for the othercompound groups above. The subgroups in group 24 are 24-1 through 24-6,24-7-1 through 24-7-6, 24-8-1 through 24-8-6, 24-9-1 through 24-9-6 andso on essentially as described for the groups above.

Group 24. Group 24 comprises each compound named in compound groups 1through 23 where R³ moieties 1-10 listed in Table A are replaced withthe following moieties:

-   1 —O—C(S)—O—C1-C8 alkyl-(OH)₀₋₂-   2 —O—C(O)—O—C1-C8 alkyl-(OH)₀₋₂-   3 —C(O)—C1-C6 alkyl-O—C1-C2 alkyl-   4 —C(O)—C1-C6 alkyl-(S)₀₋₁—C1-C2 alkyl-(OH)₀₋₁-   5 —C(O)—C1-06 alkyl-NH₀₋₂(C1-C4 alkyl)₀₋₂-   6 —O—C(O)—C4-C4 alkyl-heterocycle-   7 —C(O)—O—C1-C4 alkyl-C6H₃₋₅—(OH)₀₋₂-   8 —O—C(O)—O—C1-C4 alkyl-C6H₃₋₅—(OH)₀₋₂-   9 —O—C(O)—C1-C4 alkyl-C6H₃₋₅—(O—C1-C4 alkyl)₀₋₂-   10 —O—C(O)—C1-C4 alkyl-C6H₃₋₅-(halogen)₀₋₂

Group 24 comprises subgroups 23-1 through 23-21-17-10-8-6, which namecompounds or genera of compounds essentially as described for the othercompound groups above. The 1728 subgroups in group 23 are 23-1 through23-6, 23-7-1 through 23-7-6, 23-8-1 through 23-8-6, 23-9-1 through23-9-6 and so on essentially as described for the groups above.

Group 25. Group 25 comprises each compound named in compound groups 1through 23, but where R³ moieties 1-10 listed in Table A are replacedwith the following moieties:

-   1 —O—C₀₋₄ alkyl-heterocycle-   2 —O—C(O)—C₀₋₄ alkyl-heterocycle-   3 —SH-   4 ═S-   5 —C₂-C₆ alkyl-(OH)₁₋₂-   6 —O—CHR²⁴—C(O)—R²⁵-   7 —O—CHR²⁴—C(O)—N(R²⁵)₂-   8 —O—CHR²⁴—C(O)—NHR²⁵-   9 —O—CHR²⁴—C(O)—NH₂-   10 —O—CHR²⁴—C(O)—OC₆H₅

Group 25 comprises subgroups 25-1 through 25-23-21-17-10-8-6, which namecompounds or genera of compounds essentially as described for the othercompound groups above. The subgroups in group 25 are 25-1 through 25-6,25-7-1 through 25-7-6, 25-8-1 through 25-8-6, 25-9-1 through 25-9-6 andso on essentially as described for the groups above.

Group 26. Group 26 comprises each compound or genus named in compoundgroups 1 through 25, but wherein R¹ is not divalent, i.e., it is notbonded to the carbon atom at the 3 position by a double bond (e.g., R¹is not ═O) and it is in the α-configuration, instead of theβ-configuration as shown in formula B.

Group 26 comprises subgroups 26-1 through 26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups above. The subgroups in group 26 are 26-1 through26-6, 26-7-1 through 26-7-6, 26-8-1 through 26-8-6, 26-9-1 through26-9-6 and so on essentially as described for the groups above.

Group 27. Group 27 comprises each compound or genus named in compoundgroups 1 through 26, but wherein R² is not divalent, i.e., it is notbonded to the carbon atom at the 3 position by a double bond (e.g., R²is not ═O) and it is in the α-configuration, instead of theβ-configuration as shown in formula B.

Group 27 comprises subgroups 27-1 through 27-26-25-23-21-17-10-8-6,which name compounds or genera of compounds essentially as described forthe other compound groups above. The subgroups in group 27 are 27-1through 27-6, 27-7-1 through 27-7-6, 27-8-1 through 27-8-6, 27-9-1through 27-9-6 and so on essentially as described for the groups above.

Group 28. Group 28 comprises each compound or genus named in compoundgroups 1 through 27, but wherein R³ is not divalent, i.e., it is notbonded to the carbon atom at the 3 position by a double bond (e.g., R³is not ═O) and it is in the β-configuration, instead of theα-configuration as shown in formula B.

Group 28 comprises subgroups 28-1 through 28-27-26-25-23-21-17-10-8-6,which name compounds or genera of compounds essentially as described forthe other compound groups above. The subgroups in group 28 are 28-1through 28-6, 28-7-1 through 28-7-6, 28-8-1 through 28-8-6, 28-9-1through 28-9-6 and so on essentially as described for the groups above.

Group 29. Group 29 comprises each compound or genus named in compoundgroups 1 through 28, but wherein R⁴ is not divalent, i.e., it is notbonded to the carbon atom at the 3 position by a double bond (e.g., R⁴is not ═O) and it is in the α-configuration, instead of theβ-configuration as shown in formula B.

Group 29 comprises subgroups 29-1 through29-28-27-26-25-23-21-17-10-8-6, which name compounds or genera ofcompounds essentially as described for the other compound groups above.The subgroups in group 29 are 29-1 through 29-6, 29-7-1 through 29-7-6,29-8-1 through 29-8-6, 29-9-1 through 29-9-6 and so on essentially asdescribed for the groups above.

Group 30. Group 30 comprises each compound or genus named in compoundgroups 1 through 29, but wherein R⁵ is in the α-configuration, insteadof the 3-configuration as shown in formula B.

Group 30 comprises subgroups 30-1 through30-29-28-27-26-25-23-21-17-10-8-6, which name compounds or genera ofcompounds essentially as described for the other compound groups above.The subgroups in group 30 are 30-1 through 30-6, 30-7-1 through 30-7-6,30-8-1 through 30-8-6, 30-9-1 through 30-9-6 and so on essentially asdescribed for the groups above.

Group 31. Group 31 comprises each compound or genus named in compoundgroups 1 through 30, but wherein R⁵ is in the α-configuration, insteadof the β-configuration as shown in formula B.

Group 31 comprises subgroups 31-1 through31-30-29-28-27-26-25-23-21-17-10-8-6, which name compounds or genera ofcompounds essentially as described for the other compound groups above.The subgroups in group 31 are 31-1 through 31-6, 31-7-1 through 31-7-6,31-8-1 through 31-8-6, 31-9-1 through 31-9-6 and so on essentially asdescribed for the groups above.

Group 32. Group 32 comprises each compound or genus named in compoundgroups 1 through 31, but wherein the hydrogen atom at the 5 position isin the β-configuration, instead of the α-configuration as shown informula B.

Group 32 comprises subgroups 32-1 through32-31-30-29-28-27-26-25-23-21-17-10-8-6, which name compounds or generaof compounds essentially as described for the other compound groupsabove. The subgroups in group 32 are 32-1 through 32-6, 32-7-1 through32-7-6, 32-8-1 through 32-8-6, 32-9-1 through 32-9-6 and so onessentially as described for the groups above.

Group 33. Group 33 comprises each compound or genus named in compoundgroups 1 through 32, but wherein the hydrogen atom at the 8 position isin the α-configuration, instead of the β-configuration as shown informula B.

Group 33 comprises subgroups 33-1 through33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, which name compounds orgenera of compounds essentially as described for the other compoundgroups above. The subgroups in group 33 are 33-1 through 33-6, 33-7-1through 33-7-6, 33-8-1 through 33-8-6, 33-9-1 through 33-9-6 and so onessentially as described for the groups above.

Group 34. Group 34 comprises each compound or genus named in compoundgroups 1 through 33, but wherein the hydrogen atom at the 9 position isin the β-configuration, instead of the α-configuration as shown informula B.

Group 34 comprises subgroups 34-1 through34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, which name compounds orgenera of compounds essentially as described for the other compoundgroups above. The subgroups in group 34 are 34-1 through 34-6, 34-7-1through 34-7-6, 34-8-1 through 34-8-6, 34-9-1 through 34-9-6 and so onessentially as described for the groups above.

Group 35. Group 35 comprises each compound or genus named in compoundgroups 1 through 34, but wherein the hydrogen atom at the 14 position isin the β-configuration, instead of the α-configuration as shown informula B.

Group 35 comprises subgroups 35-1 through35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, which name compoundsor genera of compounds essentially as described for the other compoundgroups above. The subgroups in group 35 are 35-1 through 35-6, 35-7-1through 35-7-6, 35-8-1 through 35-8-6, 35-9-1 through 35-9-6 and so onessentially as described for the groups above.

Group 36. Group 36 comprises each compound or genus named in compoundgroups 1 through 35, but wherein R⁴ in formula B is not divalent, and asecond monovalent R⁴ is present at the 17 position, and the second R⁴ isa moiety other than hydrogen. As used here, monovalent R⁴ means that thesecond R⁴ moiety is bonded to the carbon atom at the 17 position by asingle bond.

The second R⁴ optionally comprises —OH, —OR^(PR), —SH, —SR^(PR), —NH₂,—NHR^(PR), a halogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl, optionally substituted alkylaryl, an optionallysubstituted heterocycle, an ester, an ether, a thioester, a thionoester,a thioether, an optionally substituted monosaccharide, an optionallysubstituted oligosaccharide, a carbonate, a carbamate, an amide or anamino acid. Any of these moieties, may comprise any R⁴ structuredisclosed herein.

Exemplary second R⁴ moieties include —C≡C—(CH₂)_(n)H (e.g., —C≡CH and—C≡C—CH₃), —C═C—(CH₂)_(n)H, —(CH₂)_(n)H (e.g., —CH₃, —C₂H₅, —C₃H₇),—(CH₂)_(n)C₆H₅, wherein n is 0, 1, 2, 3, 4, 5, 6, 7 or 8 and any ofthese exemplary second R⁴ moieties optionally comprise 1, 2, 3, 4 ormore independently selected —O—, —OH, ═O, —S—, —SH, ═S, —NH—, —NH₂,—COOH, —COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN, —NO₂, ═NHO, —CH₃, —CF₃,—C₂H₅ or —C₆H₅ moieties that replace (or substitute) one or morehydrogen or carbon atoms, wherein such moieties may be adjacent to oneanother, e.g., they can comprise —C(O)—NH— or —NH—C(O)—NH—. Typicallymoieties that replace a hydrogen or carbon atom will not replace adivalent or trivalent carbon atom, e.g., in —CH═CH— or in —C≡C— andspecific embodiments include one or more substitutions at carbons thatare separated from a —CH═CH— or —C≡C— moiety by one, two, three or more—CH₂-moieties. In some embodiments, one or two hydrogen atoms that arebonded to the distal carbon atom is substituted by one or two —OH,═O—SH, ═S, —NH₂, —COOH, —COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN, —NO₂ or═NHO moieties.

Group 36 comprises subgroups 36-1 through36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, which namecompounds or genera of compounds essentially as described for the othercompound groups above. The subgroups in group 36 are 36-1 through 36-6,36-7-1 through 36-7-6, 36-8-1 through 36-8-6, 36-9-1 through 36-9-6 andso on essentially as described for the groups above.

Group 37. Group 37 comprises each compound or genus named in compoundgroups 1 through 36, but wherein R⁷ in formula B is not —CH₂— or aheteroatom, i.e., R¹⁰ is bonded to R⁷ in formula B and it is not ahydrogen atom.

The R¹⁰ optionally comprises —OH, ═O, —OR^(PR), —SH, ═S, —SR^(PR), —NH₂,—NHR^(PR), a halogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl, optionally substituted alkylaryl, an optionallysubstituted heterocycle, an ester, an ether, a thioester, a thionoester,a thioether, an optionally substituted monosaccharide, an optionallysubstituted oligosaccharide, a carbonate, a carbamate, an amide or anamino acid. Any of these moieties, may comprise any R¹⁰ structuredisclosed herein.

Exemplary second R⁴ moieties include —C≡C—(CH₂)_(n)H (e.g., —C≡CH and—C≡C—CH₃), —C═C—(CH₂)_(n)H, —(CH₂)_(n)H (e.g., —CH₃, —C₂H₅, —C₃H₇),—(CH₂)_(n)C₆H₅, wherein n is 0, 1, 2, 3, 4, 5, 6, 7 or 8 and any ofthese exemplary second R⁴ moieties optionally comprise 1, 2, 3, 4 ormore independently selected —O—, —OH, ═O, —S—, —SH, ═S, —NH—, —NH₂,—COOH, —COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN, —NO₂, ═NHO, —CH₃, —CF₃,—C₂H₅ or —C₆H₅ moieties that replace (or substitute) one or morehydrogen or carbon atoms, wherein such moieties may be adjacent to oneanother, e.g., they can comprise —C(O)—NH— or —NH—C(O)—NH—. In someembodiments, the moieties that replace a hydrogen or carbon atom willnot replace a divalent or trivalent carbon atom, or a hydrogen that isbonded to such a carbon atom, e.g., in —CH═CH— or in —C═C— and specificembodiments include one or more substitutions at carbons that areseparated from a —CH═CH— or —C≡C— moiety by one, two, three or more—CH₂— moieties. In some embodiments, one or two hydrogen atoms that arebonded to the distal carbon atom is substituted by one, two or three—OH, ═O—SH, ═S, —NH₂, —COOH, —COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN,—NO₂ or ═NHO moieties.

Group 37 comprises subgroups 37-1 through37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, which namecompounds or genera of compounds essentially as described for the othercompound groups above. The subgroups in group 37 are 37-1 through 37-6,37-7-1 through 37-7-6, 37-8-1 through 37-8-6, 37-9-1 through 37-9-6 andso on essentially as described for the groups above.

Group 38. Group 38 comprises each compound or genus named in compoundgroups 1 through 37, but wherein R⁸ in formula B is not —CH₂— or aheteroatom, i.e., R¹⁰ is bonded to R⁸ in formula B and it is not ahydrogen atom.

This R¹⁰ optionally comprises —OH, ═O, —OR^(PR), —SH, ═S, —SR^(PR),—NH₂, —NHR^(PR), a halogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl, optionally substituted alkylaryl, an optionallysubstituted heterocycle, an ester, an ether, a thioester, a thionoester,a thioether, an optionally substituted monosaccharide, an optionallysubstituted oligosaccharide, a carbonate, a carbamate, an amide or anamino acid. Any of these moieties, may comprise any R¹⁰ structuredisclosed herein.

Other exemplary R¹⁰ moieties include —C≡C—(CH₂)_(n)H (e.g., —C≡CH and—C≡C—CH₃), —C═C—(CH₂)_(n)H, —(CH₂)_(n)H (e.g., —CH₃, —C₂H₅, —C₃H₇),—(CH₂)_(n)C₆H₅, wherein n is 0, 1, 2, 3, 4, 5, 6, 7 or 8 and any ofthese exemplary second R⁴ moieties optionally comprise 1, 2, 3, 4 ormore independently selected —O—, —OH, ═O, —S—, —SH, ═S, —NH—, —NH₂,—COOH, —COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN, —NO₂, ═NHO, —CH₃, —CF₃,—C₂H₅ or —C₆H₅ moieties that replace (or substitute) one or morehydrogen or carbon atoms, wherein such moieties may be adjacent to oneanother, e.g., they can comprise —C(O)—NH— or —NH—C(O)—NH—. In someembodiments, moieties that replace a hydrogen or carbon atom will notreplace a divalent or trivalent carbon atom, or a hydrogen that isbonded to such a carbon atom, e.g., in —CH═CH— or in —C═C— and specificembodiments include one or more substitutions at carbons that areseparated from a —CH═CH— or —C≡C— moiety by one, two, three or more—CH₂— moieties. In some embodiments, one or two hydrogen atoms that arebonded to the distal carbon atom is substituted by one, two or three—OH, ═O—SH, ═S, —NH₂, —COOH, —COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN,—NO₂ or ═NHO moieties.

Group 38 comprises subgroups 38-1 through38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, which namecompounds or genera of compounds essentially as described for the othercompound groups above. The subgroups in group 38 are 38-1 through 38-6,38-7-1 through 38-7-6, 38-8-1 through 38-8-6, 38-9-1 through 38-9-6 andso on essentially as described for the groups above.

Group 39. Group 39 comprises each compound or genus named in compoundgroups 1 through 38, but wherein R⁹ in formula B is not —CH₂— or aheteroatom, i.e., R¹⁰ is bonded to R⁹ in formula B and it is not ahydrogen atom and wherein when a double bond is present at the 1-2position, this R¹⁰ is not bonded to R⁹ by a double bond. Thus, thecarbon atom at the 2 position is not pentavalent or charged.

This R¹⁰ optionally comprises —OH, ═O, —OR^(PR), —SH, ═S, —SR^(PR),—NH₂, —NHR^(PR), a halogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl, optionally substituted alkylaryl, an optionallysubstituted heterocycle, an ester, an ether, a thioester, a thionoester,a thioether, an optionally substituted monosaccharide, an optionallysubstituted oligosaccharide, a carbonate, a carbamate, an amide or anamino acid. Any of these moieties, may comprise any R¹⁰ structuredisclosed herein.

Other exemplary R¹⁰ moieties include —C≡C—(CH₂)_(n)H (e.g., —C≡H and—C≡C—CH₃), —C═C—(CH₂)_(n)H, —(CH₂)_(n)H (e.g., —CH₃, —C₂H₅,—(CH₂)_(n)C₆H₅, wherein n is 0, 1, 2, 3, 4, 5, 6, 7 or 8 and any ofthese exemplary second R⁴ moieties optionally comprise 1, 2, 3, 4 ormore independently selected —O—, —OH, ═O, —S—, —SH, ═S, —NH—, —NH₂,—COOH, —COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN, —NO₂, ═NHO, —CH₃, —CF₃,—C₂H₅ or —C₆H₅ moieties that replace (or substitute) one or morehydrogen or carbon atoms, wherein such moieties may be adjacent to oneanother, e.g., they can comprise —C(O)—NH— or —NH—C(O)—NH—. In someembodiments the moieties that replace a hydrogen or carbon atom will notreplace a divalent or trivalent carbon atom, or a hydrogen that isbonded to such a carbon atom, e.g., in —CH═CH— or in —C═C— and specificembodiments include one or more substitutions at carbons that areseparated from a —CH═CH— or —C≡C— moiety by one, two, three or more—CH₂— moieties. In some embodiments, one or two hydrogen atoms that arebonded to the distal carbon atom is substituted by one, two or three—OH, ═O—SH, ═S, —NH₂, —COOH, —COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN,—NO₂ or ═NHO moieties.

Group 39 comprises subgroups 39-1 through39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, which namecompounds or genera of compounds essentially as described for the othercompound groups above. The subgroups in group 39 are 39-1 through 39-6,39-7-1 through 39-7-6, 39-8-1 through 39-8-6, 39-9-1 through 39-9-6 andso on essentially as described for the groups above.

Group 40. Group 40 comprises each compound or genus named in compoundgroups 1 through 39, wherein R⁷ in formula B is —O—, instead of a —CH₂—or —CHR¹⁰— moiety, where R¹⁰ is not hydrogen. Group 40 comprisessubgroups 40-1 through40-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups. The subgroups in group 40 are 40-1 through 40-6,40-7-1 through 40-7-6, 40-8-1 through 40-8-6, 40-9-1 through 40-9-6 andso on essentially as described for the groups above. The subgroup 40-1,40-2 40-8-1, 40-8-2, 40-11-1 and 40-11-2 compounds named 1.2.5.9 havethe structures

Subgroup 40-8-1 and 40-8-2 compounds named 1.2.5.9 have the structures

The subgroup 40-11-1 and 40-11-2 compounds named 1.2.5.9 have thestructures

Group 41. Group 41 comprises each compound or genus named in compoundgroups 1 through 39, wherein R⁸ in formula B is —O—, instead of a —CH₂—or —CHR¹⁰— moiety, where R¹⁰ is not hydrogen. Group 41 comprisessubgroups 41-1 through41-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups. The subgroups in group 41 are 41-1 through 41-6,41-7-1 through 41-7-6, 41-8-1 through 41-8-6, 41-9-1 through 41-9-6 andso on essentially as described for the groups above. Group 41 compoundsare named in essentially the same manner as described for group 40 andother compound groups. Thus, for example, subgroup 41-1, 41-2, 41-8-1,41-8-2, 41-11-1 and 41-11-2 compounds named 1.2.5.9 have the structuresshown for these compounds in group 40, except that an oxygen atom ispresent at the 11 position and no oxygen is present at the position.

Group 42. Group 42 comprises each compound or genus named in compoundgroups 1 through 39, wherein R⁸ in formula B is —O—, instead of a —CH₂—or —CHR¹⁰— moiety, where R¹⁰ is not hydrogen. Group 42 comprisessubgroups 42-1 through42-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups. The subgroups in group 42 are 42-1 through 42-6,42-7-1 through 42-7-6, 42-8-1 through 42-8-6, 42-9-1 through 42-9-6 andso on essentially as described for the groups above. Group 42 compoundsare named in essentially the same manner as described for group 40 andother compound groups. Thus, for example, subgroup 42-1, 42-2, 42-8-1,42-8-2, 42-11-1 and 42-11-2 compounds named 1.2.5.9 have the structuresshown for these compounds in group 40, except that an oxygen atom ispresent at the 2 position and no oxygen is present at the position.

This group does not include species or genera of compounds wherein adouble bond is present at the 1-2 position, since this would make theoxygen atom charged. Therefore, there is, e.g., no group 42-3, 42-4,42-6, 42-7-3, 42-7-4 or 42-7-6, since the 3, 4 and 6 groups and theirvariants all have a double bond at the 1-2 position.

Group 43. Group 43 comprises each compound or genus named in compoundgroups 1 through 39, wherein R⁷ in formula B is —NH—, instead of a —CH₂—or —CHR¹⁰— moiety, where R¹⁰ is not hydrogen. Group 43 comprisessubgroups 43-1 through43-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups. The subgroups in group 43 are 43-1 through 43-6,43-7-1 through 43-7-6, 43-8-1 through 43-8-6, 43-9-1 through 43-9-6 andso on essentially as described for the groups above. The subgroup 43-1,43-2 43-8-1, 43-8-2, 43-11-1 and 43-11-2 compounds named 1.2.5.9 havethe structures shown for these compounds in group 40, except that —NH—is present at the 15 position instead of oxygen.

Group 44. Group 44 comprises each compound or genus named in compoundgroups 1 through 39, wherein R⁸ in formula B is —NH—, instead of a —CH₂—or —CHR¹⁰— moiety, where R¹⁰ is not hydrogen. Group 44 comprisessubgroups 44-1 through44-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups. The subgroups in group 44 are 44-1 through 44-6,44-7-1 through 44-7-6, 44-8-1 through 44-8-6, 44-9-1 through 44-9-6 andso on essentially as described for the groups above. Group 44 compoundsare named in essentially the same manner as described for group 40 andother compound groups. Thus, for example, subgroup 44-1, 44-2, 44-8-1,44-8-2, 44-11-1 and 44-11-2 compounds named 1.2.5.9 have the structuresshown for these compounds in group 40, except that —NH— is present atthe 11 position and no oxygen is present at the 15 position.

Group 45. Group 45 comprises each compound or genus named in compoundgroups 1 through 39, wherein R⁹ in formula B is —NH— or —N═, instead ofa —CH₂— or —CHR¹⁰— moiety, where R¹⁰ is not hydrogen. Group 45 comprisessubgroups 45-1 through45-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups. The subgroups in group 45 are 45-1 through 45-6,45-7-1 through 45-7-6, 45-8-1 through 45-8-6, 45-9-1 through 45-9-6 andso on essentially as described for the groups above. Group 45 compoundsare named in essentially the same manner as described for group 40 andother compound groups. Thus, for example, subgroup 45-1, 45-2, 45-8-1,45-8-2, 45-11-1 and 45-11-2 compounds named 1.2.5.9 have the structuresshown for these compounds in group 40, except that —NH— is present atthe 2 position and no oxygen is present at the 15 position.

Group 46. Group 46 comprises each compound or genus named in compoundgroups 1 through 39, wherein R⁷ in formula B is —S—, instead of a —CH₂—or —CHR¹⁰— moiety, where R¹⁰ is not hydrogen. Group 46 comprisessubgroups 46-1 through46-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups. The subgroups in group 46 are 46-1 through 46-6,46-7-1 through 46-7-6, 46-8-1 through 46-8-6, 46-9-1 through 46-9-6 andso on essentially as described for the groups above. The subgroup 46-1,46-2 46-8-1, 46-8-2, 46-11-1 and 46-11-2 compounds named 1.2.5.9 havethe structures shown for these compounds in group 40, except that —S— ispresent at the 15 position instead of oxygen.

Group 47. Group 47 comprises each compound or genus named in compoundgroups 1 through 39, wherein R⁸ in formula B is —S—, instead of a —CH₂—or —CHR¹⁰— moiety, where R¹⁰ is not hydrogen. Group 47 comprisessubgroups 47-1 through47-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups. The subgroups in group 47 are 47-1 through 47-6,47-7-1 through 47-7-6, 47-8-1 through 47-8-6, 47-9-1 through 47-9-6 andso on essentially as described for the groups above. Group 47 compoundsare named in essentially the same manner as described for group 40 andother compound groups. Thus, for example, subgroup 47-1, 47-2, 47-8-1,47-8-2, 47-11-1 and 47-11-2 compounds named 1.2.5.9 have the structuresshown for these compounds in group 40, except that —S— is present at the11 position and no oxygen is present at the 15 position.

Group 48. Group 48 comprises each compound or genus named in compoundgroups 1 through 39, wherein R⁹ in formula B is —S—, instead of a —CH₂—or —CHR¹⁰— moiety, where R¹⁰ is not hydrogen. Group 48 comprisessubgroups 48-1 through48-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6, whichname compounds or genera of compounds essentially as described for theother compound groups. The subgroups in group 48 are 48-1 through 48-6,48-7-1 through 48-7-6, 48-8-1 through 48-8-6, 48-9-1 through 48-9-6 andso on essentially as described for the groups above. Group 48 compoundsare named in essentially the same manner as described for group 40 andother compound groups. Thus, for example, subgroup 48-1, 48-2, 48-8-1,48-8-2, 48-11-1 and 48-11-2 compounds named 1.2.5.9 have the structuresshown for these compounds in group 40, except that —S— is present at the2 position and no oxygen is present at the 15 position.

This group does not include species or genera of compounds wherein adouble bond is present at the 1-2 position. Therefore, there is, e.g.,no group 48-3, 48-4, 48-6, 48-7-3, 48-7-4 or 48-7-6, since the 3, 4 and6 groups and their variants all have a double bond at the 1-2 position.

Group 49. Group 49 comprises each compound or genus named in compoundgroups 1 through 39, but wherein two of R⁷, R⁸ and R⁹ in formula Bindependently are —O—, —NH—, ═NH— or —S—, instead of —CH₂— or —CHR¹⁰—,where R¹⁰ is not hydrogen. This group includes 27 combinations of twoheteroatoms (O, N or S) that are at any two of R⁷, R⁸ and R⁹. These are(49c1, i.e., combination number 1) O2-O11 (i.e., oxygen at the 2 and 11positions), (49c2) O2-O15, (49c3) O11-O15, (49c4) O2-N11 (i.e., oxygenat the 2-position and nitrogen at the 11 position), (49c5) O2-N15,(49c6) O11-N15, (49c7) O2-S11 (i.e., oxygen at the 2-position and sulfurat the 11 position), (49c8) O2-S15, (49c9) O11-S15, (49c10) N2-N11,(49c11) N2-N15, (49c12) N11-N15, (49c13) N2-O11, (49c14) N2-O15, (49c15)N11-O15, (49c16) N2-S11, (49c17) N2-S15, (49c18) N11-S15, (49c19)S2-S11, (49c20) S2-S15, (49c21) S11-S15, (49c22) S2-O11, (49c23) S2-O15,(49c24) S11-O15, (49c25) S2-N11, (49c26) S2-N15 and (49c27) S11-N15.

Group 49 comprises subgroups 49c1-1 through49c27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6,which name compounds or genera of compounds essentially as described forthe other compound groups. The subgroups in group 49 are 49c1-1 through49c1-6, 49c1-7-1 through 49c1-7-6, 49c1-8-1 through 49c1-8-6, 49c1-9-1through 49c1-9-6 and so on essentially as described for the groupsabove. Group 49 compounds are named in essentially the same manner asdescribed for group 40 and other compound groups. Thus, for example,subgroup 49c1-1, 49c1-2, 49c1-8-1, 49c1-8-2, 49c1-11-1 and 49c1-11-2compounds named 1.2.5.9 have the structures shown for these compounds ingroup 40, except that —O— is present at the 2 and 11 positions and nooxygen is present at the 15 position. Similarly, subgroup 49c10-1,49c10-2, 49c10-8-1, 49c10-8-2, 49c10-11-1 and 49c10-11-2 compounds named1.2.5.9 have the structures shown for these compounds in group 40,except that —NH— or ═N— is present at the 2 and 11 positions and nooxygen is present at the 15 position. This group does not includespecies or genera of compounds wherein a double bond and either —O— or—S— is present at the 2-position.

Group 50. Group 50 comprises each compound or genus named in compoundgroups 1 through 39, but wherein all three of R⁷, R⁸ and R⁹ in formula Bindependently are —O—, —NH—, ═NH— or —S—, instead of —CH₂— or —CHR¹⁰—,where R¹⁰ is not hydrogen. This group includes all combinations of 3heteroatoms (O, N or S) that are at R⁷, R⁸ and R⁹. The combinations aredefined essentially as described for the combinations in group 49. Theyare (50c1) O2-O11-O15, (50c2) O2-O11-N15, (50c3) O2-N11-O15, (50c4)O2-N11-N15, (50c5) O2-O11-S15, (50c6) O2-S11-O15, (50c7) O2-S11-S15,(50c8) N2-N11-N15, (50c9) N2-N11-O15, (50c10) N2-O11-N15, (50c11)N2-O11-O15, (50c12) N2-N11-S15, (50c13) N2-S11-N15, (50c14) N2-S11-S15,(50c15) S2-S11-S15, (50c16) S2-S11-O15, (50c17) S2-O11-S15, (50c18)S2-S11-O15, (50c19) S2-S11-N15, (50c20) S2-N11-S15, (50c21) S2-N11-N15,(50c22) S2-N11-S15, (50c23) O2-S11-N15, (50c24) N2-O11-S15, (50c25)N2-S11-O15, (50c26) S2-O11-N15 and (50c27) S2-N11-O15.

Group 50 comprises subgroups 50c1-1 through50c27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6,which name compounds or genera of compounds essentially as described forthe other compound groups. The subgroups in group 50 are 50c1-1 through50c1-6, 50c1-7-1 through 50c1-7-6, 50c1-8-1 through 50c1-8-6, 50c1-9-1through 50c1-9-6 and so on essentially as described for the groupsabove. Group 50 compounds are named in essentially the same manner asdescribed for group 40 and other compound groups. Thus, for example,subgroup 50c1-1, 50c1-2, 50c1-8-1, 50c1-8-2, 50c1-11-1 and 50c1-11-2compounds named 1.2.5.9 have the structures shown for these compounds ingroup 40, except that —O— is also present at the 2 and 11 positions.Similarly, subgroup 50c10-1, 50c10-2, 50c10-8-1, 50c10-8-2, 50c10-11-1and 50c10-11-2 compounds named 1.2.5.9 have the structures shown forthese compounds in group 40, except that —NH— or ═N— is present at the 2and 15 positions and oxygen is present at the 11 position. This groupdoes not include species or genera of compounds wherein a double bondand either —O— or —S— is present at the 2-position.

Group 51. Group 51 comprises each compound or genus named in compoundgroups 1 through 50, but wherein R⁷ comprises a —X—CHR¹⁰— moiety, whereX is —O—, —NR^(PR)— or —S—. This group includes all R⁷ moieties, i.e.,(51a1) —O—CHR¹⁰—, (51a2) —NR^(PR)—CHR¹⁰—, (51a3) —S—CHR¹⁰—, (51a4)—CHR¹⁰—O—, (51a5) —CHR¹⁰—NR^(PR)— and (51a6) —CHR¹⁰—S—. Group 51comprises subgroups 51a1-1 through51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6,which name compounds or genera of compounds essentially as described forthe other compound groups. The subgroups in group 51 are 51a1-1 through51a1-6, 51a1-7-1 through 51a1-7-6, 51a1-8-1 through 51a1-8-6, 5a1-9-1through 51a1-9-6 and so on essentially as described for the groupsabove.

In some embodiments, the R¹⁰ included in R⁷ is hydrogen. In others, theR¹⁰ included in R⁷ is optionally comprises —OH, ═O, —OR^(PR), —SH, ═S,—SR^(PR), —NH₂, —NHR^(PR), a halogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted alkylaryl, anoptionally substituted heterocycle, an ester, an ether, a thioester, athionoester, a thioether, an optionally substituted monosaccharide, anoptionally substituted oligosaccharide, a carbonate, a carbamate, anamide or an amino acid. The R¹⁰ may comprise any R¹⁰ structure disclosedherein.

Other exemplary R¹⁰ moieties include —C≡C—(CH₂)_(n)H (e.g., —C≡H and—C≡C—CH₃), —C═C—(CH₂)_(n)H, —(CH₂)_(n)H (e.g., —CH₃, —C₂H₅,—(CH₂)_(n)C₆H₅, wherein n is 0, 1, 2, 3, 4, 5, 6, 7 or 8 and any ofthese exemplary R¹⁰ moieties optionally comprise 1, 2, 3, 4 or moreindependently selected —O—, —OH, ═O, —S—, —SH, ═S, —NH—, —NH₂, —COOH,—COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN, —NO₂, ═NHO, —CH₃, —CF₃, —C₂H₅or —C₆H₅ moieties that replace (or substitute) one or more hydrogen orcarbon atoms, wherein such moieties may be adjacent to one another,e.g., they can comprise —C(O)—NH— or —NH—C(O)—NH—. In some embodimentsthe moieties that replace a hydrogen or carbon atom will not replace adivalent or trivalent carbon atom, or a hydrogen that is bonded to sucha carbon atom, e.g., in —CH═CH— or in —C≡C— and specific embodimentsinclude one or more substitutions at carbons that are separated from a—CH═CH— or —C≡C— moiety by one, two, three or more —CH₂— moieties. Insome embodiments, one or two hydrogen atoms that are bonded to thedistal carbon atom is substituted by one, two or three —OH, ═O—SH, ═S,—NH₂, —COOH, —COOR^(PR), —F, —Cl, —Br, —I, —SCN, —CN, —NO₂ or ═NHOmoieties.

Group 52. Group 52 comprises each compound or genus named in compoundgroups 1 through 49, but wherein R⁷ is absent and the ring in formula Bthat contains R⁷ comprises a cyclobutyl moiety with R³ and one or two R⁴bonded to it. Group 52 comprises subgroups 52-1 through52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6,which name compounds or genera of compounds essentially as described forthe other compound groups. The subgroups in group 52 are 52-1 through52-6, 52-7-1 through 52-7-6, 52-8-1 through 52-8-6, 52-9-1 through52-9-6 and so on essentially as described for the groups above.

Group 53. Group 53 comprises each compound or genus named in compoundgroups 1 through 52, but wherein R⁸ is absent and the ring in formula Bthat contains R⁸ comprises a 5 membered ring moiety. Group 53 comprisessubgroups 53-1 through53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6,which name compounds or genera of compounds essentially as described forthe other compound groups. The subgroups in group 53 are 53-1 through53-6, 53-7-1 through 53-7-6, 53-8-1 through 53-8-6, 53-9-1 through53-9-6 and so on essentially as described for the groups above.

The subgroups here do not include compounds or genera where two ringheteroatoms are present as described in group 49 and where both R⁷ andR⁸ are absent (“group 53-52-49- . . . ”), since such groups are mutuallyincompatible. This holds for all of the compound groups describedherein, i.e., whenever the structures that a first group or subgroupspecifies is incompatible with the structure that a second group orsubgroup specifies, then the structure that the first group or subgroupspecifies is not included. However, all other possible compounds andgenera are included in such compound groups.

Group 54. Group 54 comprises each compound or genus named in compoundgroups 1 through 53, but wherein R⁹ is absent and the ring in formula Bthat contains R⁹ comprises a 5 membered ring moiety. Group 54 comprisessubgroups 54-1 through54-53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6,which name compounds or genera of compounds essentially as described forthe other compound groups. The subgroups in group 54 are 54-1 through54-6, 54-7-1 through 54-7-6, 54-8-1 through 54-8-6, 54-9-1 through54-9-6 and so on essentially as described for the groups above. Thesubgroups here do not include, e.g., compounds or genera where two orthree ring heteroatoms are present as described in group 49 or 50 andwhere two or three of R⁷, R⁸ and R⁹ are absent (e.g., “group 54-52-49- .. . ”).

The individual compounds and genera named in groups 1-54 above may alsobe named using any suitable formal or informal chemical nomenclature.Thus, as will be apparent, individual compounds in these groups include16α-bromoepiandrosterone, 16α-hydroxyepiandrosterone,3α,16α-dihydroxy-5α-androstane-17-one,3α,16α,17β-trihydroxy-5α-androstane,3α,16α,17α-trihydroxy-5α-androstane, 3β,17β-dihydroxyandrost-5-ene or3β,7β,17β-trihydroxyandrost-5-ene, 7-oxodehydroepiandrosterone,16α-fluoroandrost-5-ene-17-one,7α-hydroxy-16α-fluoroandrost-5-ene-17-one,7β-hydroxy-16α-fluoroandrost-5-ene-17-one,17α-hydroxy-16α-fluoroandrost-5-ene, 17β-hydroxy-16α-fluoroandrost-5-eneand the like.

Any of the species of compounds or genera of compounds that aredisclosed herein, e.g., as named in compound groups 1 through54-53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6or elsewhere in this disclosure, are suitable for use in the methods asdescribed herein or in the cited references.

Additional embodiments of the formula 1 compounds include any compoundor genus of compounds that are disclosed herein, e.g., any of thecompounds or genera of compounds in groups 1 through 54 wherein one orboth of R⁵ or R⁶ independently comprises —CH₂SH, —CHO, —CH₂NRPR,—CH₂NH₂, —C₂H₅, —C₂H₄OH, —C₂H₄SH, —C₂H₄NH₂, —CH₂CHO, —CH₂CH₂NR^(PR),—CH₂CH₂OH, —CH₂CH₂SH, —CH₂CH₂C₆H₅, —CH₂C₆H₅ or —C₆H₅ wherein any phenyl(C₆H₅) moiety in the foregoing groups is optionally substituted at thephenyl ring with 1, 2, 3, 4 or 5 moieties independently selected fromthose described for esters herein and including C₁₋₆ alkyl (optionallysubstituted with 1 or 2 independently selected —OH, —SH, —O—, —S— or—NH—) C1-6 alkoxy, —F, —Cl, —Br, —I, —CN, —NO₂, —OH, —SH, —COOR^(PR),—NHR^(PR) and —C(O)—C1-6 alkyl.

In some embodiments, one or more of the variable groups that are bondedto the formula 1 compound, e.g., R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸,independently have the structure(s) and/or independently comprise thenamed compounds, —H, —OH, ═O, —SH, ═S, —NH₂, —CN, —N₃, halogen, —CHO,—CHS, ═CH₂, ═NOH, ═NOC(O)CH₃, —C(O)—CH₃, —C(O)—(CH₂)₁₄—CH₃, —C≡H,—C≡CH₃, —CH═CH₂, —CH═CH₂CH₃, —O—C(O)—(CH₂)_(m)—(CF₂)_(n)—CH₃, —O—C(O)—(CH₂)_(m)—(CF₂)_(n)—CF₃, —O—C(O)—(CH₂)_(m)—(CF₂)_(n)—CH₂F,—O—C(O)—O—(CH₂)_(m)—(CF₂)_(n)—CH₃, —O—C(O)—O—(CH₂)_(m)—(CF₂)_(n)—CF₃,—O—C(O)—O—(CH₂)_(m)—(CF₂)_(n)—CH₂F, —O—C(O)—NH—(CH₂)_(m)—(CF₂)_(n)—CH₃,C(O)—NH—(CH₂)_(m)—(CF₂)_(n)—CF₃, —O—C(O)—NH—(CH₂)_(m)—(CF₂)_(n)—CH₂F(where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n is 0, 1, 2, 3, 4, 5, 6,7, 8, 9 or 10, usually n is 0), —CH(CH₃)—(CH₂)₂—C(O)NH—CH₂COOH,—CH(CH₃)—(CH₂)₂—C(O)NH—CH₂SO₃H, —OSi(CH₃)₂C(CH₃)₃, —C(OH)═CHCH₃,═CH(CH₂)₀₋₁₅CH₃, —(CH₂)₀₋₁₄CH₂F, —(CH₂)₀₋₁₄CH₂Cl, —(CH₂)₀₋₁₄CH₂Br,—(CH₂)₀₋₁₄CH₂I, —(CH₂)₂₋₁₀—O—(CH₂)₀₋₄—CH₃, —(CH₂)₂₋₁₀—S—(CH₂)₀₋₄—CH₃,—(CH₂)₂₋₁₀—NH—(CH₂)₀₋₄—CH₃, —O—(CH₂)₀₋₁₄CH₂F, (CH₂)₀₋₁₄CH₂Cl,—O—(CH₂)₀₋₁₄CH₂Br, —O—(CH₂)₀₋₁₄CH₂ ¹, —O—(CH₂)₂₋₁₀—(CH₂)₀₋₄—CH₃,—O—(CH₂)₂₋₁₀—S—(CH₂)₀₋₄CH₃, —O—(CH₂)₂₋₁₀—NH—(CH₂)₀₋₄CH₃,—O—C(O)—(CH₂)₀₋₁₄CH₂F, —O—C(O)—(CH₂)₀₋₁₄CH₂Cl, —O—C(O)—(CH₂)₀₋₁₄CH₂Br,—O—C(O)—(CH₂)₀₋₁₄CH₂I, —O—C(O)—(CH₂)₂₋₁₀—O—(CH₂)₀₋₄O—H₃,—O—C(O)—(CH₂)₂₋₁₀—S—(CH₂)₃₋₄—CH₃, —O—C(O)—(CH₂)₂₋₁₀—NH—(CH₂)₃₋₄CH₃,—O—C(S)—(CH₂)₀₋₁₄CH₂F, —O—C(S)—(CH₂)₀₋₁₄CH₂Cl, —O—C(S)—(CH₂)₀₋₁₄CH₂Br,—O—C(S)—(CH₂)₀₋₁₄CH₂I, —O—C(S)—(CH₂)₂₋₁₀—O—(CH₂)₀₋₄CH₃,—O—C(S)—(CH₂)₂₋₁₀—S—(CH₂)₀₋₄—CH₃, —O—C(S)—(CH₂)₂₋₁₀—NH—(CH₂)₀₋₄—CH₃,—(CH₂)₀₋₁₆NH₂, —(CH₂)₀₋₁₅CH₃, —(CH₂)₀₋₁₅CN, —(CH₂)₀₋₁₅CH═CH₂,—(CH₂)₀₋₁₅NHCH(O), —(CH₂)₀₋₁₆NH—(CH₂)₀₋₁₅CH₃, —(CH₂)₀₋₁₅CCH,—(CH₂)₀₋₁₅OC(O)CH₃, —(CH₂)₀₋₁₅OCH(OH)CH₃, —(CH₂)₀₋₁₅C(O)OCH₃,—(CH₂)₀₋₁₅C(O)OCH₂CH₃, —(CH₂)₀₋₁₅C(O)(CH₂)₀₋₁₅CH₃,—(CH₂)₀₋₁₅C(O)(CH₂)₀₋₁₅CH₂OH, —O(CH₂)₁₋₁₆NH₂, —O(CH₂)₁₋₁₅CH₃,—O(CH₂)₁₋₁₅CN, —O(CH₂)₁₋₁₅CH═CH₂, —O(CH₂)₁₋₁₅NHCH(O),—O(CH₂)₁₋₁₆NH—(CH₂)₁₋₁₅CH₃, —O(CH₂)₁₋₁₅CCH, —O(CH₂)₁₋₁₅C(O)CH₃,—O(CH₂)₁₋₁₅OCH(OH)CH₃, —O(CH₂)₁₋₁₅C(O)OCH₃, —O(CH₂)₁₋₁₅C(O)OCH₂CH₃,—O(CH₂)₁₋₁₅C(O)(CH₂)₀₋₁₅CH₃, —O(CH₂)₁₋₁₅C(O)(CH₂)₀₋₁₅CH₂OH,—OC(O)(CH₂)₁₋₁₆NH₂, —OC(O)(CH₂)₁₋₁₅CH₃, —C(O)O(CH₂)₁₋₁₅CN,—C(O)O(CH₂)₁₋₁₅CH═CH₂, —OC(O)(CH₂)₁₋₁₅NHCH(O),—OC(O)(CH₂)₁₋₁₆NH—(CH₂)₁₋₁₅CH₃, —OC(O)(CH₂)₁₋₁₅CCH,—OC(O)(CH₂)₁₋₁₅OC(O)CH₃, —OC(O)(CH₂)₁₋₁₅OCH(OH)CH₃,—OC(O)(CH₂)₁₋₁₅C(O)OCH₃, —OC(O)(CH₂)₁₋₁₅C(O)OCH₂CH₃,—OC(O)(CH₂)₁₋₁₅C(O)(CH₂)₀₋₁₅CH₃, —OC(O)(CH₂)₁₋₁₅C(O)(CH₂)₀₋₁₅CH₂OH,phosphoenolpyruvate, D-glucosamine, glucholic acid, glucuronic acid,pantothenic acid, pyruvic acid, glucose, fructose, mannose, sucrose,lactose, fucose, rhamnose, galactose, ribose,(O-1)-D-galactopyranosyl-(1-O-4)-D-glucopyranoside,(O-1)-tetra-O-acetyl-D-glucopyranosyl-(1-O-4)-tri-O-acetyl-D-glucopyranoside,2′-deoxyribose, 3′-deoxyribose, glycerol, 3-phosphoglycerate, a PEG (PEG20, PEG 100, PEG 200, PEG 10000), a polyoxyalkylene polymer, glycine,alanine, phenylalanine, threonine, proline, 4-hydroxyproline or anoligonucleotide or analog that comprises about 4 to about 21 monomers.

In some embodiments, an R³ and an R⁴ of the formula 1 compoundscomprises a ring(s) structure. Exemplary compounds of formula 2 includethe following structures,

wherein, R¹⁶ independently are —CH₂—, —O—, —S— or —NH—; R¹⁵, R¹⁷ and R¹⁸independently are —H, —OR^(PR), —SR^(PR), —N(R^(PR))₂, —O—Si—(R¹³)₃,—CHO, —CHS, —CH═NH, —CN, —SCN, —NO₂, —OSO₃H, —OPO₃H, an ester, athioester, a thionoester, a phosphoester, a phosphothioester, aphosphonoester, a sulfite ester, a sulfate ester, an amide, an aminoacid, a peptide, an ether, a thioether, an acyl group, a thioacyl group,a carbonate, a carbamate, an acetal, a thioacetal, a halogen, anoptionally substituted alkyl group, an optionally substituted alkenylgroup, an optionally substituted alkynyl group, an optionallysubstituted aryl moiety, an optionally substituted heteroaryl moiety, anoptionally substituted heterocycle, an optionally substitutedmonosaccharide, an optionally substituted oligosaccharide, a nucleoside,a nucleotide, an oligonucleotide, a polymer, or, one or more of R¹⁵, R¹⁷and R¹⁸ independently are ═O, ═S, ═NOH or ═CH₂ and the hydrogen atomthat is bonded to the same carbon atom is absent; and R¹⁹ is nitrogen orCH.

Such compounds include any of these structures wherein one, two or threeof R⁷, R⁸ and R⁹ are independently —O—, —S—, or —NH— or wherein one orboth of R⁵ and R⁶ independently are —H, —CH₃, —CH₂OR^(PR), —CH₂OH,—CH₂SH, —CH₂SR^(PR), —CH₂O—C(O)—C₁₋₁₀ alkyl, —CH₂S—C(O)—C₁₋₁₀ alkyl,—CH₂O—C(O)—C₁₋₁₀ alkenyl, —CH₂S—C(O)—C₁₋₁₀ alkenyl, —CH₂O—C(O)—C₀₋₄alkyl-heterocycle, —CH₂S—C(O)—C₀₋₄ alkyl-heterocycle, —CH₂O—C(O)—C₀₋₄alkyl-phenyl, —CH₂S—C(O)—C₀₋₄ alkyl-phenyl, wherein any C₁₋₁₀ alkyl,heterocycle or phenyl moiety is optionally substituted with one or moresubstituents, wherein the one or more substituents are one, two, threeor more independently selected —O—, ═O, —OR^(PR), —S—, ═S, —SR^(PR),—NH—, —N(R^(PR))₂ or —C(O)—NH—, wherein each R^(PR) independently is —Hor a protecting group.

Exemplary formula 1 compounds will comprise compounds where the steroidhas the structure

and (1) one of the atoms or groups described immediately above at the 1,2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 16 or 17 positions, withindependently selected groups at the remaining variable group positions,e.g., —H, —OH, ═O, —SH, —CHO, —CH₂—, —O—, —S—, —NH—, halogen, optionallysubstituted alkyl, acyl, ester or any other moiety described herein, (2)two of these groups, which are the same or are independently selectedand are at, e.g., the 2,3, 2,7, 2,11, 2,15, 2,16, 2,17, 3,7, 3,11, 3,15,3,16, 3,17, 7,11, 7,15, 7,16, 7,17, 11,15, 11,16, 11,17 or 16,17positions, with independently selected groups at the remaining variablegroup positions, e.g., —H, —OH, ═O, —SH, —CHO, —CH₂—, —O—, —S—, —NH—,halogen, optionally substituted alkyl, acyl, ester or any other moietydescribed herein, (3) three of these groups, which are the same or areindependently selected and are at, e.g., the 2,3,7, 2,3,11, 2,3,15,2,3,16, 2,3,17, 3,7,11, 3,7,15, 3,7,16, 3,7,17, 7,11,15, 7,11,16,7,11,17, 11,15,16, 11,15,17, or 15,16,17 positions, with independentlyselected groups at the remaining variable group positions, e.g., —H,—OH, ═O, —SH, —CHO, —CH₂—, —O—, —S—, —NH—, halogen, optionallysubstituted alkyl, acyl, ester or any other moiety described herein, (4)four of these groups, which are the same or are independently selectedand are at, e.g., the 2,3,7,11, 2,3,7,15, 2,3,7,16, 2,3,7,17, 3,7,11,15,3,7,11,16, 3,7,11,17, 7,11,15,16, 7,11,15,17 or 11,15,16,17 positions,with independently selected groups at the remaining variable grouppositions, e.g., —H, —OH, ═O, —SH, —CHO, —CH₂—, —O—, —S—, —NH—, halogen,optionally substituted alkyl, acyl, ester or any other moiety describedherein or (5) any of the foregoing compounds in (1) through (4) wherein1, 2 or 3 R¹⁰ at the 1, 4, 5, 6, 9, 12 and 14 positions are —OH, —SH,halogen, an ester, a thioester, a thionoester, optionally substitutedalkyl (e.g., C1-C8), optionally substituted alkoxy (e.g., C1-C8),optionally substituted alkenyl (e.g., C2-C8), or an optionallysubstituted heterocycle or any other moiety described herein and theremaining R¹⁰ are —H.

When a substituent is an oligonucleotide or a polymer usually only a oneof these is bonded to the formula 1 compound. Typically, when R¹-R² andR⁴-R⁶ comprise one or more of these substituents (or others describedherein), the substituent is present in the β-configuration, while R³typically comprises a substituent in the β-configuration. In someembodiments, R² is in the α-configuration.

In some embodiments, one or more of the variable groups that are bondedto the formula 1 compounds, e.g., R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸,independently comprise a nucleoside, a nucleotide, an oligonucleotide oran analog of any of these moieties. Typically such moieties are linkedto the steroid nucleus through a terminal hydroxyl, thiol, acyl moietyor amine at the 5′, 3′ or 2′ positions, when a hydroxyl, thiol, acylmoiety or amine is present at that position. For oligonucleotides andoligonucleotide analogs, the linkage to the steroid occasionally isthrough a sugar hydroxyl at an internal 2′ position.

Analogs of phosphodiester linkages include phosphorothioate linkages andothers as described in the cited references. Oligonucleotide couplinggroups means any moiety suitable for generating a phosphodiester linkageor phosphodiester analog linkage between adjacent nucleotides or theiranalogs. Suitable oligonucleotide coupling groups include —OH,H-phosphonate, alkylphosphonamidites or phosphoramidites such asβ-cyanoethyl-phosphoramidite,N,N-diisopropylamino-β-cyanoethoxyphosphine and others as described inthe cited references. Suitable purine and pyrimidine bases includeadenine, guanine, cytosine, thymine, uracil and others as described inthe cited references. Suitable nucleosides, nucleotides,oligonucleotides and their analogs have been described, see e.g., U.S.Pat. Nos. 4,725,677, 4,973,679, 4,997,927, 4,415,732, 4,458,066,5,047,524, 4,959,463, 5,212,295, 5,386,023, 5,489,677, 5,594,121,5,614,622, 5624621; and PCT publication Nos. WO 92/07864, WO 96/29337,WO 97/14706, WO 97/14709, WO 97/31009, WO 98/04585 and WO 98/04575 allof which are incorporated herein by reference. The formula 1 compounds,e.g., species or genera named in any of the compound groups 1 through54-53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6,are suitable for linkage to oligonucleotides modulate the lipophilicityof oligonucleotides or the transport or permeation of an oligonucleotideinto cells. Such linkages may be biologically labile to facilitaterelease of the steroid from the oligonucleotide once the conjugate hasentered the cell.

Individual formula 1 compounds, e.g., those named in any of the compoundgroups 1 through 54 are suitable for use as standards for variousanalytical methods, e.g., for use in HPLC, MS, NMR, IR or otheranalytical methods. Thus, to aid in the determination of, e.g., thestructure of a metabolite of a formula 1 compound or a structurallyrelated compound, another structurally related formula 1 compound couldbe used. Metabolism of formula 1 compounds will include one or more ofhydroxylation or conjugation, usually to a —OH moiety, with a moietysuch as sulfate, phosphate or a monosaccharide such as glucuronic acidat, e.g., the 2, 3, 7, 11, 15, 16 or 17 positions. In these embodiments,the appropriate use of a formula 1 compound of known structure as astandard can aid in or verify the identification of metabolites that areprojected to have closely related structures. Information regarding theidentification can be useful or sometimes is necessary for, e.g.,obtaining regulatory approval to market a therapeutic agent such as aformula 1 compound or understanding the potential biological role that aformula 1 compound or its metabolite can play in one of the applicationsdisclosed herein or in a cited reference. To facilitate such uses, theformula 1 compound may be labeled as appropriate, e.g., using a formula1 compound with, e.g., a ¹³C atom at 1, 2 or more of the 1, 2, 3, 4, 6,7, 11, 12, 15, 16, 17, 18 or 19 positions in the steroid.

Table 2 shows these and other exemplary moieties that one or more ofR¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸ independently can comprise. Pr means aprotecting group. These moieties are often bonded to one or more of theR¹, R² and R⁴ positions, usually to one or two of those positions. Forstructures with more than one of a given variable, e.g., X in structureA3 or A5, each is independently selected.

TABLE 2

Typical containers for storage of compositions and formulations thatcomprise a formula 1 compound will limit the amount of water thatreaches the materials contained therein. Typically, formulations arepackaged in hermetically or induction sealed containers. The containersare usually induction sealed. Water permeation characteristics ofcontainers have been described, e.g., Containers—Permeation, chapter,USP 23 <671>, United States Pharmacopeial Convention, Inc., 12601Twinbrook Parkway, Rockville, Md. 20852, pp.: 1787 et seq. (1995).

The use of formula A compounds for treatment of certain diseases, e.g.,infections such as malaria, HCV or Cryptosporidium, has been described.Formula A compounds have the structure

where Q₁ is —C(R₁)₂— or —C(O)—; Q₂ is —C(R₁)₂—, —C(R₁)(Y)—, —C(Y)— or—CH₂—CH₂—; Q₃ is —H or —C(R₁)₃—; Q₄ is —C(R₁)₂—, —C(O)—,hydroxyvinylidine (—CH(CH═CHOH)—) or methyl methylene (—CH(CH)₃—); Q₅ is—C(R₁)₂— or —C(O)—; X and Y independently are —OH, —H, lower alkyl(e.g., C₁₋₆ alkyl), —O—C(O)—R₅, —C(O)—OR₅, halogen (i.e., —F, —Cl, —Bror —I) or ═O; each R₁ independently is —H, —F, —Cl, —Br, —I, —OH, C₁₋₆alkoxy, or C₁₋₆ alkyl; R₂ is —H, —OH, —F, —Cl, —Br, —I, C₁₋₆ alkyl, C₁₋₆alkoxy, —OR₃, an ester (e.g., —O—C(O)—R₄ or —C(O)—O—R₄), a thionoester(e.g., —O—C(S)—R₄ or —C(S)—O—R₄), a thioacetal (e.g., —S—C(O)—R₄, or—C(O)—S—R₄), a sulfate ester (e.g., —O—S(O)(O)—O—R₄), a sulfonate ester(e.g., —O—S(O)—O—R₄) or a carbamate (e.g., —O—C(O)—NH—R₄ or—NH—C(O)—O—R₄) or R₂, together with the R₁ that is bonded to the samecarbon atom is ═O; R₃ is —S(O)(O)—OM,—S(O)(O)—O—CH₂—CH(O—C(O)—R₆)—CH₂—C(O)—R₆,—P(O)(O)—O—CH₂—CH(O—C(O)—R₇)—CH₂—O—C(O)—R₇, a glucuronide group ofstructure (B)

or R₃ is C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, a C₁₋₁₈ ester or aC₁₋₁₈ thioester, a C₁₋₁₈ thionoester, where any of the foregoing C₁₋₁₈or C₂₋₁₈ moieties are optionally substituted at one or more hydrogenatoms with one or more independently selected —OR^(PR), (including —OH),—NHR^(PR), (including —NH₂) or —SR^(PR), (including —SH) groups, or R₃is a C₁₋₁₈ fatty acid, C₂₋₁₀ alkynyl, (J)_(n)-phenyl-C₁₋₆-alkyl,(J)_(n)-phenyl-C₂₋₆-alkenyl; R₄ is —H, a protecting group, optionallysubstituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈ alkenyl,optionally substituted C₁₋₁₈ alkynyl, optionally substituted aryl,optionally substituted aryl-C₁₋₆ alkyl, optionally substituted aryl-C₂₋₆alkenyl, optionally substituted aryl-C₂₋₆ alkynyl, optionallysubstituted heterocycle-C₁₋₆ alkyl, optionally substituted C₂₋₆alkenyl-heterocycle, optionally substituted C₂₋₆ alkynyl-heterocycle oran optionally substituted heterocycle, where any of the foregoingmoieties are optionally substituted at one, two, three, four, five ormore carbon or hydrogen atoms with one or more independently selected—O—, —S—, —NR^(PR)— (including —NH—), —NH—C(O)—, —OR^(PR) (including—OH), —NHR^(PR) (including —NH₂), —SR^(PR) (including —SH), ═O, ═S,═N—OH, ═CH₂, —CN, —SCN, —NO₂, —F, —Cl, —Br or —I groups or atoms; eachR₅ independently is straight or branched C₁₋₁₄ alkyl; each R₆independently is straight or branched C₁₋₁₄ alkyl; each R₇ independentlyis straight or branched C₁₋₁₄ alkyl or a glucuronide group of structure(B); each R^(PR) independently is —H or an independently selectedprotecting group; n is 0, 1, 2 or 3; each J independently is —F, —Cl,—Br, —I, C₁₋₄ alkyl, C₁₋₄ alkenyl, C₁₋₄ alkoxy, carboxy, nitro, sulfate,sulfonyl, a C₁₋₆ carboxyl ester or a C₁₋₆ sulfate ester; M is hydrogen,sodium, —S(O)(O)—O—CH₂—CH(O—C(O)—R₆)—CH₂—O—C(O)—R₆,—P(O)(O)—O—CH₂—CH(O—C(O)—R₇)—CH₂—O—C(O)—R₇ or a glucuronide group ofstructure (A); the dotted lines in formula 1 represent an optionaldouble bond, provided that there are not double bonds at both the 4-5and 5-6 positions and provided that when a double bond is present, zeroor 1 R₁ group is bonded to carbon atoms at the 1-, 2-, 4-, 5-, 6- or 17positions so that these carbon atoms are tetravalent; and the salts,stereoisomers, positional isomers, metabolites, analogs or precursors.

The formula A compounds, including compounds where both R₁ at the11-position are not hydroxyl, alkoxy or a moiety that can hydrolyze to ahydroxyl, are generally suitable for use in the methods and compositionsthat are disclosed herein, e.g., their use to enhance a subject's Th1immune responses or to treat inflammation. Methods of administration anddosages for these compounds are essentially as described herein.

The invention provides compounds having the formula V

or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof,

wherein (a) R₁ and R₂ are each independently selected from the groupconsisting of a hydrogen atom and a glucuronide group having the formula

wherein (i) R₇ is an alkyl ester wherein the alkyl is optionallysubstituted, and (ii) R₉, R₉ and R₁₀ are each —OR₁₄, wherein R₁₄ is ahydrogen atom or a protected hydroxy; and (iii) at least one of R₁ or R₂is not hydrogen; (b) R₅ and R₆ are each independently selected from thegroup consisting of a hydrogen atom, optionally substituted alkyl,hydroxy, and a protected hydroxy; or R₅ and R₆ taken together form anoxygen atom, which, together with the carbon atom to which R₅ and R₆ arejoined, forms a ketone group; and (c) R₁₂ and R₁₃ are each independentlyselected from the group consisting of a hydrogen atom, optionallysubstituted alkyl (e.g., methyl, ethyl or —CHO), hydroxy, and aprotected hydroxy. Such compounds include ones wherein (1) the protectedhydroxy is an ester such as an acetate or proprionate, (2) one of R₁ andR₂ is a hydrogen atom and the other one of R₁ and R₂ is saidglucuronide, (3) R₅ and R₆ are each independently selected from thegroup consisting of a hydrogen atom, hydroxy, and acetate, e.g., one ofR₅ and R₆ is a hydrogen atom and the other is acetate, (4) R₁₂ and R₁₃are methyl, and/or (5) R₇ is a methyl ester. These compounds includeones where R₁ is a glucuronide group, R₂ is a hydrogen atom, and R₅ andR₆ together are ═O and methyl2,3,4-trihydroxy-1-O-(7,17-dioxoandrost-5-ene-3β-yl)-β-D-glucopyranosiduronateand methyl2,3,4-tri-O-acetyl-1-O-(7,17-dioxoandrost-5-ene-3β-yl)-β-D-glucopyranosid-uronate,or a pharmaceutically acceptable salt, ester, ether, amide, or prodrugthereof.

Other compounds have the formula VI

or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof,

wherein (a) R₅ and R₆ are each independently selected from the groupconsisting of a hydrogen atom and a glucuronide group having the formula

wherein (i) R₇ is an alkyl ester wherein the alkyl is optionallysubstituted, and (ii) R₈, R₉ and R₁₀ are each —OR₁₄, wherein R₁₄ is ahydrogen atom or a protected hydroxy; and (iii) at least one of R₅ andR₆ is not hydrogen; (b) R₁₁ is a hydrogen atom or a protected hydroxy;and (c) R₁₂ and R₁₃ are each independently selected from the groupconsisting of a hydrogen atom, optionally substituted alkyl, hydroxy,and a protected hydroxy. Such compounds include ones wherein (1) theprotected hydroxy may be an ester, e.g., acetate or proprionate, (2) oneof R₅ and R₆ is a hydrogen atom and the other one of R₅ and R₆ is aglucuronide, (3) R₅ is a glucuronide group and R₆ is a hydrogen atom,(4) R₁₂ and R₁₃ are methyl, and/or (5) R₇ is methyl ester. Thesecompounds includemethyl-2,3,4-trihydroxy-1-O-(3β-αcetoxyandrost-5-ene-7-oxo-17β-yl)-β-D-gluco-pyranosiduronateandmethyl-2,3,4-tri-β-acetyl-1-O-(3β-αcetoxyandrost-5-ene-7-oxo-17β-yl)-β-D-glucopyranosiduronate,or a pharmaceutically acceptable salt, ester, ether, amide, or prodrugthereof.

Additional compounds include VII

or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof,

wherein (a) R₃ and R₄ are each independently selected from the groupconsisting of a hydrogen atom and a glucuronide group having the formula

wherein (i) R₇ is an alkyl ester wherein the alkyl is optionallysubstituted, and (ii) R₈, R₉ and R₁₀ are each —OR₁₄, wherein R₁₄ is ahydrogen atom, optionally substituted alkyl, cycloalkyl, e.g., C3, 4, 5,6, 7, or 8 cycloalkyl, or a protected hydroxy; and (iii) at least one ofR₃ and R₄ is not hydrogen; (b) R₅ and R₆ are each independently selectedfrom the group consisting of a hydrogen atom, optionally substitutedalkyl, hydroxy, and a protected hydroxy; or R₅ and R₆ taken togetherform an oxygen atom, which, together with the carbon atom to which R₅and R₆ are joined, forms a ketone group; (c) R₁₁ is a hydrogen atom or aprotected hydroxy; and (d) R₁₂ and R₁₃ are each independently selectedfrom the group consisting of a hydrogen atom, optionally substitutedalkyl, hydroxy, and a protected hydroxy. Such compounds include oneswherein (1) the protected hydroxy may be an ester, e.g., acetate orproprionate, (2) R₃ and R₄ are —H and a glucuronide, (3) R₅ and R₆ areeach independently selected from the group consisting of —H, hydroxy,and acetoxy, e.g., they are —H and acetoxy, (4) R₁₂ and R₁₃ are methyland/or R₇ is methyl ester. Such compounds include ones wherein R₃ is aglucuronide group, R₄ is —H, R₅ is acetate, or wherein R₆ is —H andwherein R₃ is —H, R₄ is a glucuronide group, and R₅ and R₆ together form═O, andmethyl-2,3,4-tri-O-acetyl-1-O-(3β,17β-diacetoxyandrost-5-ene-7β-yl)-β-D-gluco-pyranosiduronate,methyl1-O-(3β,17β-diacetoxyandrost-5-ene-7β-yl)-β-D-gluco-pyranosiduronate,andmethyl-2,3,4-tri-O-acetyl-1-O-(3β-acetoxy-17-oxoandrost-5-ene-7α-yl)-β-D-glucopyranosiduronate,or a pharmaceutically acceptable salt, ester, ether, amide, or prodrugthereof.

The compounds also include compounds having the formula VIII

or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof,

wherein (a) R₁ and R₂ are each independently selected from the groupconsisting of a hydrogen atom and —OR₁₄, wherein (i) R₁₄ is selectedfrom the group consisting of a hydrogen atom, optionally substitutedalkyl, and a protected hydroxy; and (ii) at least one of R₁ or R₂ is nothydrogen; (b) R₅, R₆, R₇, and R₈ are each independently selected fromthe group consisting of a hydrogen atom, optionally substituted alkyl,hydroxy, and a protected hydroxy; or R₅ and R₆ taken together form anoxygen atom, which, together with the carbon atom to which R₅ and R₆ arejoined, forms a ketone group; or R₇ and R₈ taken together form an oxygenatom, which, together with the carbon atom to which R₇ and R₈ arejoined, forms a ketone group; and (c) R₁₂ and R₁₃ are each independentlyselected from the group consisting of a hydrogen atom, alkyl, hydroxy,and a protected hydroxy. Such compounds include ones wherein (1) theprotected hydroxy is an ester, e.g., acetate or proprionate, or atrialkylsilyl, e.g., t-butyldimethylsilyl, and wherein R₁ and R₂ are —Hand —OR₁₄ where R₁₄ is optionally selected from methyl, ethyl, n-propyl,i-propyl, n-butyl, sec-butyl, t-butyl, pentyl, hexyl, n-octyl,n-dodecyl, 1-ethoxyethyl, t-butyldimethylsilyl, tetrahydropyran-2-yl,and —C(O)CH₃, (2) R₅ and R₆ are each independently selected from thegroup consisting of —H, —OH, and trialkylsilyl, e.g., R₅ and R₆ are —Hand a trialkylsilyl or R₅ and R₆ are —H and —OH or R₅ and R₆ togetherare ═O, (3) R₁₂ and R₁₃ are methyl, and/or (4) R₇ and R₈ areindependently —H, —OH or trialkylsilyl, e.g., R₇ and R₈ are —H and atrialkylsilyl or R₇ and R₈ are —H and —OH or R₇ and R₈ together are ═O.These compounds include 3β-tosyloxyandrost-5-ene-17-one,3β-methoxyandrost-5-ene-17-one, 3β-methoxyandrost-5-ene-7,17-dione,3β-methoxy-17,17-ethylenedioxyandrost-5-ene-7-one,3β-methoxy-17,17-ethylenedioxyandrost-5-ene-7β-ol,3β-methoxy-17,17-ethylenedioxyandrost-5-ene-7α-ol,3β-methoxyandrost-5-ene-7β,17β-diol,3β-acetoxy-7α-bromoandrost-5-ene-17-one,3β-acetoxy-7-methoxyandrost-5-ene-17-one,3β-methoxyandrost-5-ene-17β-ol,3β-methoxy-17β-hydroxyandrost-5-ene-7-one,3β-methoxy-17β-acetoxyandrost-5-ene-7-one,3β-t-butoxyandrost-5-ene-17-one, 3β-t-butoxyandrost-5-ene-7,17-dione,3β-t-butyldimethylsilyloxyandrost-5-ene-7,17-dione,3β,17β-di(t-butyldimethylsilyloxy)androst-5-ene-7-one,3β-acetoxyandrost-5-ene-7β,17β-diol,3β-acetoxyandrost-5-ene-7β,17β-di(t-butyldimethylsilyl) ether,3β-acetoxy-17β-t-butyldimethylsilyloxyandrost-5-ene-7-one,3β-(2-tetrahydropyranoxy)androst-5-ene-7,17-dione,3β-dodecanoxyandrost-5-ene-17-one,3β-dodecanoxyandrost-5-ene-7,17-dione,3β-(1′-ethoxy)ethoxyandrost-5-ene-17-one, and3β-(1′-ethoxy)ethoxyandrost-5-ene-7,17-dione, or the pharmaceuticallyacceptable salt, ester, ether, amide, or prodrug thereof.

Other compounds have the formula IX

or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof,

wherein (a) R₁ and R₂ are each independently selected from the groupconsisting of a hydrogen atom and —O—C(O)—OR₁₄, wherein (i) R₁₄ isselected from the group consisting of a hydrogen atom, optionallysubstituted alkyl, and a carbocyclic ring; and (ii) at least one of R₁or R₂ is not hydrogen; (b) R₅, R₆, R₇, and R₈ are each independentlyselected from the group consisting of a hydrogen atom, optionallysubstituted alkyl, hydroxy, —O—C(O)—OR₁₄, and a protected hydroxy; or R₅and R₆ taken together form an oxygen atom, which, together with thecarbon atom to which R₅ and R₆ are joined is ═O (a ketone); or R₇ and R₈taken together form an oxygen atom, which, together with the carbon atomto which R₇ and R₈ are joined is ═O; and (c) R₁₂ and R₁₃ are eachindependently selected from the group consisting of a hydrogen atom,alkyl, hydroxy, and a protected hydroxy. Such compounds include oneswherein (1) the protected hydroxy is an ester, e.g., acetate orproprionate, (2) R₁ and R₂ are —H and —O—C(O)—OR₁₄ and R₁₄ optionally ismethyl, ethyl, propyl, n-butyl, sec-butyl, t-butyl, n-octyl, n-dodecyl,1-ethoxyethyl, 9-fluorenylmethyl or —C(O)CH₃, (3) R₅ and R₆independently are —H, —OH, —O—C(O)—OCH₃, —O—C(O)—OC₂H₅, —O—C(O)—OC₃H₇,—O—C(O)—OC₄H₉, —O—C(O)—OCH₂C₂H₃, —O—C(O)—OCH₂C₃H₅ or—O—C(O)—O—(CH₂)₂—O—C₂H₅, (3) R₅ and R₆ are —H and —OH, e.g., R₅ and R₆are —H and —OH or together are ═O, (4) R₁₂ and R₁₃ are methyl, (4) R₇and R₈ are each independently selected from the group consisting of ahydrogen atom, hydroxy, and trialkylsilyl, e.g., R₇ and R₈ are —H and—OH or together are ═O. These compounds include3β-carbomethoxyandrost-5-ene-7,17-dione,3β-carboallyloxyandrost-5-ene-7,17-dione,3β-carboethoxyandrost-5-ene-7,17-dione,3β-carboisobutoxyandrost-5-ene-7,17-dione,3β,17β-dicaromethoxyandrost-5-ene-7-one,3β-carbooctyloxyandrost-5-ene-7,17-dione,3β-carbo(9-fluorenyl)methoxyandrost-5-ene-7,17-dione,3β-carbomethoxyandrost-5-ene-7β,17β-diol,3β-carboethoxyandrost-5-ene-7β,17β-diol, and3β-carbooctyloxyandrost-5-ene-7β,17β-diol, or an pharmaceuticallyacceptable salt, ester, ether, amide, or prodrug thereof.

The compounds of formulas V, VI, VII, VIII and IX can be incorporatedinto a composition comprising the compound and an excipient, e.g., anexcipient disclosed herein. Such compositions are useful to treatsubjects having or subject to developing the diseases, conditions orsymptoms disclosed herein, e.g., obesity, diabetes, hyperlipidemia,infection, cancer, immune suppression conditions, inflammation orautoimmune conditions. The compounds can thus be used in a method oftreatment comprising administering an amount effective of one of thesecompounds to a subject (e.g., a mammal or human) to treat the disease,condition or symptom or to modulate the subject's immune system, e.g.,to enhance a Th1 immune response or to modulate a subject's weight or toslow the progression of a disease or condition.

Dosing protocols or methods. In treating any of the conditions orsymptoms disclosed herein, one can continuously (daily) orintermittently administer the formula 1 compound(s) to a subjectsuffering from or susceptible to the condition or symptom. In treating acondition such as an infection, a hyperproliferation condition, aninflammation condition or another condition disclosed herein with aformula 1 compound intermittent dosing can avoid or ameliorate some ofthe undesired aspects normally associated with discontinuous dosing.Such undesired aspects include development of resistance of a pathogensuch as a pathogen disclosed herein, e.g., a virus or bacterium such asHIV or Staphylococcus aureus or a parasite such as a Plasmodiumparasite, to the therapeutic agent, failure of the patient or subject toadhere to a daily dosing regimen or reduction of the dosages of othertherapeutic agents and/or their associated unwanted side effects ortoxicities.

In any of the continuous (daily) or intermittent dosing protocolsdescribed herein, or in treating any of the diseases, conditions orsymptoms described herein, the formula 1 compound(s) can be administeredby one or more suitable routes, e.g., oral, buccal, sublingual,intramuscular (i.m.), subcutaneous (s.c.), intravenous (i.v.),intradermal, another parenteral route or by an aerosol. The daily dosein such methods will typically comprise about 0.05 mg/kg/day to about200 mg/kg/day, or about 0.1 to about 100 mg/kg/day, including about 0.2mg/kg/day, 0.5 mg/kg/day, about 1 mg/kg/day, about 2 mg/kg/day, about 4mg/kg/day, about 6 mg/kg/day, about 10 mg/kg/day, about 20 mg/kg/day,about 40 mg/kg/day or about 100 mg/kg/day. Higher dosages, e.g., about250 mg/kg/day, about 300 mg/kg/day or about 350 mg/kg/day can also beutilized, e.g., in some veterinary applications. One can administer theformula 1 compound(s) orally using about 4 to about 60 mg/kg/day,usually about 6-30 mg/kg/day. In some embodiments, the intermittentdosing methods exclude dosing protocols that are commonly used todeliver contraceptive steroids to, e.g., human females, such as dailydosing for 21 days, followed by no dosing for 7 days. In someembodiments, the non-aqueous formulations described herein that comprisea formula 1 compound(s) are administered i.m. or s.c., while aqueousformulations that contain formula 1 compound(s) is administered by i.v.,i.m., s.c. or other parenteral routes.

Intermittent dosing embodiments include administration of a formula 1compound, e.g., orally, topically or parenterally as follows: (1) dailydosing for about 3 to about 190 days (e.g., about 3 to about 20 days),(2) no dosing of the formula 1 compound for about 4 to about 190consecutive days (e.g., about 4 to about 20 days), (3) daily dosing forabout 3 to about 190 days (e.g., about 3 to about 20 days), and (4)optionally repeating the dosing protocol 1, 2, 3, 4, 5, 6, 10, 15, 20,30 or more times. Often, the dosing of steps (1) and (3) will bemaintained for about 3-15 consecutive days, usually about 3, 4, 5 or 6consecutive days. In general, steps (1)-(3) of the dosing protocolrecited above, will be repeated at least one time, typically at least 2,3, 4, 5 or 6 times. For infections that tend to remain chronic, e.g.,HIV, HCV or other chronic virus or parasite infection, the intermittentdosing protocol is typically maintained over a relatively long timeperiod, e.g., for at least about 6 months to about 5 or more years.

In some embodiments, the number of days of continuous dosing in steps(1) and (3) is the same in each round of treatment, i.e., each timeperiod in step (1) and (3) is the same in the initial and subsequentrounds of the method. In other embodiments they differ. Thus, in someembodiments, step (1) may comprise daily dosing of about 20 mg/day toabout 1500 mg/day (e.g., about 50, 75, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350 or 400 mg/day) of a formula 1 compound for 2, 3,4, 5, 6, 7 or more consecutive days.

Then, step (2) may comprise not administering any formula 1 compound forat least about 4, 5, 6, 7, 14, 21, 28, 42, 56, 84, 98, 112 or moreconsecutive days. Step (3) could comprise daily administration of about20 mg/day to about 1500 mg/day (e.g., about 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350 or 400 mg/day) of a formula 1 compoundfor 2, 3, 4, 5, 6, 7 or more consecutive days. Steps (1) through (3) isoptionally repeated for about 1-30 or more times. On days when theformula 1 compound is administered to the subject, it may be deliveredin a single dose or in two, three or more subdoses at, e.g., about 12hour or about 8 hour time intervals. Administration of the formula 1compound would be by one or more of the routes described herein.

Exemplary embodiments comprise (a) administering a formula 1compound(s), e.g., BrEA or an ester or carbonate of BrEA, once everyother day for about 3, 5, 7, 9, 11, 13, 20 or more days, followed by (b)no dosing for about 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 70, 84, 98, 112 or more days and then (c) administering theformula 1 compound(s) at least once more on one day, e.g., administeringthe formula 1 compound(s) once every other day for about 3, 5, 7, 11,13, 20 or more days and (d) optionally repeating (a), (b) and (c) 1, 2,3, 4, 5 or 6 times or more. A subset of these embodiments comprise (a)administering a formula 1 compound(s), e.g., BrEA or an ester orcarbonate of BrEA, once every other day for about 3, 5, 7, 9, 11, 13, 20or more days, followed by (b) no dosing for at least about 7-190consecutive days, e.g., about 10-40 days, and then (c) administering theformula 1 compound(s) at least once more on one day, e.g., administeringthe formula 1 compound(s) once every other day for about 3, 5, 7, 9, 11,13, or more days and (d) optionally repeating (a), (b) and (c) 1, 2, 3,4, 5 or 6 times or more. In any of these embodiments, one can administerthe formula 1 compound(s) in 2 or 3 subdoses per day.

Other embodiments comprise (a) administering a formula 1 compound(s),e.g., BrEA or an ester or carbonate of BrEA, once every day (or in 2 or3 daily subdoses) for 3-15 or about 8-12 days, followed by (b) no dosingfor 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30, 35, 40, 45, 50, 56, 70, 84,98, 112 or more days and then (c) administering the formula 1compound(s) at least once more on one day, e.g., administering theformula 1 compound(s) once per day for about 3-15 or about 8-12consecutive days essentially as described in step (a) and (d) optionallyrepeating (a), (b) and (c) 1, 2, 3, 4, 5 or 6 times or more. A subset ofthese embodiments are (a) administering a formula 1 compound(s), e.g.,BrEA or an ester or carbonate of BrEA, once every day for about 10 days,followed by (b) no dosing for about 10-40 days and then (c)administering the formula 1 compound(s) at least once more on one day,e.g., administering the formula 1 compound(s) once per day for about 10days and (d) optionally repeating (a), (b) and (c) 1, 2, 3, 4, 5 or 6times or more. In any of these embodiments, one can administer theformula 1 compound(s) in 2 or 3 subdoses per day.

One aspect of invention intermittent dosing is monitoring the subject'sresponse to a particular dosing regimen or schedule, e.g., to anyintermittent administration method disclosed herein. For example, whiledosing a subject who has a viral infection (e.g., HCV, HIV, SIV, SHIV),one can measure the subject's or pathogen's response, e.g., ameliorationof one or more symptoms or a change in infectious particles or viral DNAor RNA in the serum or a change in an immune parameter of interest. Oncea response is observed dosing can be continued for one, two or threeadditional days, followed by discontinuing the dosing for at least oneday (at least 24 hours), usually for at least about 2, 3, 4, 5, 6, 7,14, 21, 28, 42, 56, 70, 84, 98, 112 or more days. Once the subject'sresponse shows signs of remission (e.g., a symptom begins to intensify,viral serum DNA or RNA begins to increase or an immune parameter, e.g.,as described herein, begins to deteriorate), dosing can be resumed foranother course. An aspect of the subject's response to formula 1compound(s) is that the subject may show a measurable response within ashort time, usually about 5-10 days, which allows straightforwardtracking of the subject's response, e.g., by monitoring viral titer inperipheral white blood cells (“PBMC”), by measuring viral nucleic acidlevels in the blood or by measuring a white blood cell population(s) orexpression of a cytokine or interleukin by e.g., white blood cells or asubset(s) thereof. One may monitor one or more immune cell subsets,e.g., NK, LAK, dendritic cells or cells that mediate ADCC immuneresponses, during and after intermittent dosing to monitor the subject'sresponse and to determine when further administration of the formula 1compound is indicated. These cell subsets are monitored as describedherein, e.g., by flow cytometry.

For any of the treatments or methods described herein, prolongedbeneficial effects or a sustained immune response by a subject mayresult from a single administration or a few daily administrations ofthe formula 1 compound for from intermittent treatment with the formula1 compound. A single administration means that a formula 1 compound isadministered to the subject in one, two, three or more doses within a 24hour period and no further administration of any formula 1 compound tothe subject occurs for at least about 7-90 days, e.g., about for atleast about 30 days to about 2 months, or for about 1.5, 2, 3, 4, 5, 6or more months. Prolonged beneficial effects or immune responses mayalso persist after a short course of treatment has been completed (e.g.,daily dosing for 2, 3, 4, 5 or 6 days) and the subject is no longerreceiving any formula 1 compound, or, in some cases, any othertherapeutic treatment to treat the primary cause of the subject'spathological condition. Such beneficial effects can persist for morethan about 5-30 days, e.g., for at least about 21, 28, 42, 56, 70, 84,98, 112 or more days.

Other intermittent dosing embodiments comprise administering to asubject having or susceptible to a condition as described herein aneffective amount of a formula 1 compound using an initial induction orhigh dosing regimen. The high dosing regimen may comprise, e.g., 1, 2,3, 4, 5, 6, 7 or more daily doses of about 4 to about 40 mg/kg that areadministered daily, every other day, every 3^(rd) day, every 4^(th) dayor every 5^(th) day. Then, the subject is not dosed with a formula 1compound for a period, e.g., of about 7, 14, 21, 28, 42, 56, 70, 84, 98,112 or more consecutive days. Then a lower daily dosing regimen isadministered to the subject, e.g., about 0.2 mg/kg to about 4 or about 6mg/kg, essentially as described for the high dosing regimen.Alternatively, this low dosing regimen may comprise 1, 2, 3, 6 or morerounds of a low to moderate initial level, e.g., about 2 to about 10mg/kg/day, optionally followed by subsequent rounds of daily dosing thatdecrease the initial low to moderate level by about 10%, 20%, 30%, 40%or more in each subsequent round of treatment, which is continued untiladministration is discontinued.

In some cases, beneficial effects from treatment have been observed formore than 3 months (4 or 5 or more months) after a short course oftreatment of a subject with a formula 1 compound. Thus, administrationof a formula 1 compound provides a method to help protect a subjectagainst progression of an infection or against adverse consequences ofunwanted immune reactions (e.g., inflammation) or againstimmunosuppression (from infection, chemotherapy, or as disclosedherein), without any dosing of the compound for at least 3 months afteran initial dosing protocol, which could be an intermittent or acontinuous dosing protocol over, e.g., 1 day to about 4 months (1-15days, about 1 month, about 2 months, etc).

Dosages of the formula 1 compound, routes of administration and the useof combination therapies with other standard therapeutic agents ortreatments could be applied essentially as described above for any ofthe diseases or conditions that are disclosed herein. Thus, the formula1 compounds may be administered prophylactically or therapeutically inchronic conditions or they may be administered at the time of orrelatively soon after an acute event such as the onset of surgery, amigraine or the occurrence of trauma, e.g., accidental central nervoussystem injury or a cerebral stroke or infarction. For acute events, theformula 1 compounds may thus be administered concurrently, e.g., withinabout 15 minutes or about 30 minutes of the onset or occurrence of theacute event, or at a later time, e.g., at about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 36, 42, 48,54, 60, 72, 84, 96, 108 or 120 hours after the onset or occurrence ofthe acute event. The formula 1 compounds may thus be administered atabout 6-120 hours, or about 8-48 hours, about 10-24 hours or about 12-16hours after an acute event starts or occurs.

Alternatively, the formula 1 compounds may thus be administered before,e.g., within about 15 minutes or about 30 minutes before the onset oroccurrence of a planned or anticipated acute event, or at an earliertime, e.g., at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 18, 20, 22, 24, 26, 28, 30, 36, 42, 48, 54, 60, 72, 84, 96, 108 or120 hours before the onset or occurrence of the acute event. The formula1 compounds may thus be administered at about 6-120 hours, or about 8-48hours, about 10-24 hours or about 12-16 hours before the planned oranticipated acute event starts or occurs.

Invention embodiments include a method to modulate an immune or cellularresponse in a subject in need thereof comprising administering to thesubject, or delivering to the subject's tissues, an effective amount ofa compound of formula 1. Immune and cellular response modulationincludes enhancing Th1 immune responses, reducing Th2 immune responses,reducing Th1 immune responses, enhancing Th2 immune responses, reducingunwanted or pathological inflammation, enhancing hemopoiesis ormodulating the synthesis, level or a biological activity of abiomolecule such as (1) a transcription factor such as a steroidreceptor or other associated steroid receptor factor, (2) a purine suchas adenosine, (3) a nucleotide cofactor such as NADPH, (4) a cytokine orinterleukin or a receptor for a cytokine or interleukin, or (5) anotherbiomolecule as disclosed herein. Such enhancements, reductions, levelsor activities are usually in an easily detectable range, e.g., a changecompared to a suitable control of at least about 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or a range that is between about any two ofthese values. Typically the subject is in need of such treatment, e.g.,by having a clinical condition disclosed herein or being subject todeveloping such a condition, e.g., having been exposed or potentiallyexposed to a pathogen or having a predisposing condition such asprecancer.

In some embodiments one or more formula 1 compounds or groups of formula1 compounds may excluded from one or more of the uses disclosed herein.For example, if the subject is in need of enhanced hemopoiesis, theformula 1 compound optionally excludes 5-androstene-3β-ol-17-one,5-androstene-3β,17β-diol, 5-androstene-3β,7β,17β-triol or a derivativeof any of these three compounds that can convert to these compounds byhydrolysis, or if the subject has or is susceptible to developing amemory impairing neurological disorder or memory impairment condition,the compound is not 5-androstene-3β-ol-7,17-dione or5-androstene-3β,7-diol-17-one or a derivative of these compounds thatcan has a group at the 7-position that can convert to —OH or ═O byhydrolysis. In other embodiments, formula 1 compound is not4-pregnene-11β,17α,21-triol-3,20-dione,17α,21-dihydroxypregn-4-ene-3,11,20-trione,11β,21-dihydroxy-3,20-dioxopregn-4-en-18-al,11β,17α,21-trihydroxypregna-1,4-diene-3,20-dione,17α,21-dihydroxypregna-1,4-diene-3,11,20-trione,3β-hydroxypregn-5-ene-20-one, 3β-hydroxyandrost-5-ene-17-one,pregn-4-ene-3,20-dione, 21-hydroxypregn-4-ene-3,20-dione,9-fluoro-11β,16α,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione,9-fluoro-11β,16α,17,21-tetrahydroxypregna-1,4-diene-3,20-dione,9-fluoro-11β,17α,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione,dehydroepiandrosterone-3-sulfate,1,4-pregnadiene-17α,21-diol-3,11,20-trione, androsterone, androsteroneacetate, androsterone propionate, androsterone benzoate, androstenediol,androstenediol-3-acetate, androstenediol-17-acetate,androstenediol-3,17-diacetate, androstenediol-17-benzoate,androstenediol-3-acetate-17-benzoate, androstenedione,dehydroepiandrosterone, 4-dihydrotestosterone, 5α-dihydrotestosterone,dromostanolone, dromostanolone propionate, ethylestrenol, nandrolonephenpropionate, nandrolone decanoate, nandrolone furylpropionate,nandrolone cyclohexanepropionate, nandrolone benzoate, nandrolonecyclohexanecarboxylate, oxandrolone, stanozolol, testosterone, methyltestosterone, testolactone, oxymetholone, fluoxymesterone,acetoxypregnenolone, allylestrenol, anagestone acetate, chlormadinoneacetate, cyproterone, cyproterone acetate, desogestrel,dihydrogesterone, dimethisterone, ethisterone (17α-ethynyltestosterone),ethynodiol diacetate, fluorogestone acetate, gestadene,hydroxyprogesterone, hydroxyprogesterone acetate, hydroxyprogesteronecaproate, hydroxymethylprogesterone, hydroxymethylprogesterone acetate,3-ketodesogestrel, levonorgestrel, lynestrenol, medrogestone,medroxyprogesterone acetate, megestrol, megestrol acetate, melengestrolacetate, norethindrone, norethindrone acetate, norethisterone,norethisterone acetate, norethynodrel, norgestimate, norgestrel,norgestrienone, normethisterone, and progesterone, progesterone,cyproterone acetate, norethindrone, norethindrone acetate,levonorgestrel, an ester of any of the foregoing compounds (e.g.,acetate, enanthate, propionate, isopropionate, cyclopropionate,isobutyrate, butyrate, valerate, caproate, isocaproate, hexanoate,heptanoate, octanoate, nonanoate, decanoate, undecanoate, phenylacetateor benzoate esters, e.g., hydroxyl esters), a naturally occurringglucorcorticoid, a species disclosed herein or a derivative of any ofthese that can convert to these molecules by hydrolysis or metabolism,e.g., a metabolizable or hydrolyzable ester or ether such as a cyclicketal, an acetate, a diacetete, a proprionate, a diproprionate, or anO-alkyl, an acyl, e.g., —C(O)—C1-06 alkyl or another moiety that isbonded at, e.g., a variable group such as for R¹-R⁶ as disclosed herein.

Synthesis methods. Reagents and reaction conditions that one can use tomake the formula 1 compounds have been described, see e.g., thecitations above, U.S. Pat. Nos. 5,874,598, 5,874,597, 5,874,594,5,840,900; PCT publication number WO 9901579. General chemical syntheticmethods to link a variety of organic moieties to various reactive groupshave been described. For example, in G. T. Hermanson, BioconjugateTechniques, Academic Press, 1996, functional targets such as aminoacids, peptides and carbohydrates are described at pages 3-136, whilethe chemistries of reactive groups in the functional targets, e.g.,amine, thiol, carboxyl, hydroxyl, aldehyde, ketone and reactive hydrogenatoms (e.g., —H linked to an electron-donating moiety such as aheteroaryl moiety) are described at pages 137-166. This reference alsodescribes reagents useful to make the derivatives, e.g., zero-lengthcross-linkers, heterobifunctional cross-linkers, homobifunctionalcross-linkers, tags, probes and polymers are described at pages 169-416and 605-638. This reference also describes synthetic methods to modifyoligonucleotides at pages 639-671.

In one aspect, amino acids or peptides are linked to the steroid throughthe amine group using a coupling reagent such as phosgene (Cl—CO—Cl) orCl—CS—Cl and suitably protected amino acids or peptides and steroids,which are protected as needed. Such linkage generates an intervening—CO—O— or a —CS—O— moiety between the amino acid or peptide and thesteroid nucleus.

By way of exemplification and not limitation, the following methods areused to prepare the one or more of the compounds disclosed herein.Starting materials and straightforward variations of the schemes arefound, e.g., in the following references, which are incorporated hereinby reference: A. P. Davis, et al., Tetrahedron Lett., 33: 5111-5112,1992; I. Takashi, et al., Chem. Pharm. Bull., 34: 1929-1933, 1986; I.Weisz, et al., Arch. Pharm., 319: 952-953, 1986; T. Watabe, et al., J.Med. Chem., 13: 311-312, 1970; M. Davis, et al., J. Chem. Soc. C., (11):1045-1052, 1967; R. C. Cambie, et al., J. Chem. Soc., Perkin Trans. 1,(20): 2250-2257, 1977; L. Minale, et al., J. Chem. Soc., Perkin Trans.1, (20): 2380-2384, 1974; C. K. Lai, et al., Steroids, 42: 707-711,1983; S. Irie, et al., Synthesis, (9): 1135-1138, 1996; E. J. Corey, J.Am. Chem. Soc., 118: 8765-8766, 1996; M. E. Annunziato, et al.,Bioconjugate Chem., 4: 212-218, 1993; N. J. Cussans, et al., J. Chem.Soc., Perkin Trans. 1, (8): 1650-1653, 1980; D. H. R. Barton, et al., J.Chem. Soc., Chem. Commun., (9): 393-394, 1978; H. Loibner, et al., Helv.Chim. Acta, 59: 2100-2113, 1976; T. R. Kasturi, et al., Proc. IndianAcad. Sci., [Ser.]: Chem. Sci., 90: 281-290, 1981; T. Back, J. Org.Chem., 46: 1442-1446, 1981; A. Canovas, et al., Helv. Chim. Acta, 63:486-487, 1980; R. J. Chorvat, et al., J. Org. Chem., 43: 966-972, 1978;M. Gumulka, et al., Can. J. Chem., 63: 766-772, 1985; H. Suginome, etal., J. Org. Chem., 55: 2170-2176, 1990; C. R. Engel, et al., Can.Heterocycles, 28: 905-922, 1989; H. Sugimone, et al., Bull, Chem. Soc.Jpn., 62: 193-197, 1989; V. S. Salvi, et al., Can. Steroids, 48: 47-53,1986; C. R. Engel, et al., Can. Steroids, 47: 381-399, 1986; H.Suginome, et al., Chem. Lett., (5): 783-786, 1987; T. Iwadare, et al.,J. Chem. Soc., Chem. Commun., (11): 705-706, 1985; H. Nagano, et al., J.Chem. Soc., Chem. Commun., (10): 656-657, 1985; V. S. Salvi, et al.,Steroids, 27: 717-725, 1976; C. H. Engel, et al, Steroids, 25: 781-790,1975; M. Gobbini, et al., Steroids, 61: 572-582, 1996; A. G. Gonzalez,et al., Tetrahedron, 46: 1923-1930, 1990; S. C. Bobzin, et al., J. Org.Chem., 54: 3902-3907, 1989; B. Solaja, et al., Croat. Chem. Acta, 59:1-17, 1986; Y. Kashman, et al., Tetrahedron, 27: 3437-3445, 1971; K.Yoshida, et al., Chem. Pharm. Bull. (Tokyo), 15: 1966-1978, 1967; P. B.Sollman, et al., Chem. Commun. (11): 552-554, 1967; H. Suginome, et al.,J. Org. Chem., 55: 2170-2176, 1990; H. Suginome, et al., Journal Chem.Lett., (5): 783-786, 1987; G. A. Tolstikov, et al., Zh. Org. Khim., 22:121-132, 1986; T. Terasawa, et al., J. Chem. Soc., Perkin Trans. 1, (4):990-1003, 1979; Z. Zhuang, et al., Yougi Huaxue, (4): 281-285, 1986; W.T. Smith, et al., Trans. Ky. Acad. Sci., 45: 76-77, 1984; A. K. Batta,et al., Steroids, 64: 780-784, 1999; B. Ruan, et al., Steroids, 65:29-39, 2000; L. Gamido, et al., Steroids, 65: 85-88, 2000; P. Ramesh, etal., Steroids, 64: 785-789, 1999; M. Numazawa, et al., Steroids, 64:187-196, 1999; P. N. Rao, et al., Steroids, 64: 205-212, 1999; M.Numazawa, et al., Steroids, 64: 320-327, 1999; U.S. Pat. Nos. 3,281,431,3,301,872, 3,325,535, 3,325,536, 3,952,018, 4,602,008, 5,571,795,5,627,270, 5,681,964, 5,744,453; international publication numbers WO9408588, WO 9508558, WO 9508559, WO 9638466, WO 9809450; United Kingdompatent numbers GB 1168227, GB 813529, GB 802618; French patent number824529; Japan patent number JP 45010134; European patent applications EP232788, EP 430078; and German patent number DE 19631189.

Exemplary synthesis methods are shown below.

Scheme 1. For the structures shown in scheme 1, R⁵-R⁹ are as defined forformula 1 compounds. Thus, when R⁵ and R⁶ are both —CH₃ in theβ-configuration, R⁷, R⁸ and R⁹ are all —CH₂—, H at the 9 and 14positions are in the α-configuration, acetate at the 3-position is inthe β-configuration, and H at the 8 position is in the β-configuration,the first compound in scheme 1 is DHEA acetate. The acetate groups atthe 3, 7, 16, 17 or other positions in this scheme and in other schemesdisclosed herein may independently be other ester moieties as describedherein, e.g., C₂₋₅₀ esters including —C(O)—(CH₂)₀₋₄—(CF₂)₀₋₄—CF₃,including —C(O)—CF₃, —C(O)—C₂₋₂₉ optionally substituted alkyl,—C(O)—CH₂—C₂₋₂₈ optionally substituted alkenyl, —C(O)—CH₂—C₂₋₂₈optionally substituted alkynyl, —C(O)—(CH₂)₀₋₆-optionally substitutedphenyl, or —C(O)—(CH₂)₀₋₆-optionally substituted heterocycle or otherorganic moieties as disclosed herein or in the cited references.

Typical substituents for these organic moieties are as described herein,including one, two, three or more independently selected —O—, ═O,optionally protected hydroxyl, —S—, optionally protected thiol, —NH—,optionally protected —NH₂, optionally protected —C(O)OH, —C(O)—NH—,—C(O)—NH₂, —NH₂—C(O)—H, —NH₂—C(O)—C₀₋₄H₁₋₉, —NH₂—C(O)—O—C₀₋₄H₁₋₉, —CN,—NO₂, —N₃ or halogen. Reactive groups are protected as needed, e.g., ═Owould usually be protected in the LiCR reaction that is used to generatecompound 1 in scheme 1 below.

Scheme 2. Compounds of formula 2 are prepared from structure A compoundsshown in scheme 1 using the last two steps of Scheme 1: (1a)dibromantin, (1b) LiBr, (2) Li—C≡R, where R is CR^(A) and R^(A) is asdefined above, e.g., —H, —CH₃, —CH₂N₃, —CH₂NH₂, —CH₂—O-organic moiety,—CH₂—S-organic moiety, —C₁₋₁₂ optionally substituted alkyl. When R⁷, R⁸and R⁹ are all —CH₂—, H at the 9 and 14 positions are in theα-configuration and H at the 8 position is in the β-configuration thefirst compound in scheme 1 is DHEA acetate. Typical substituents for theR^(A) alkyl moiety includes one, two or more independently selected —O—,optionally protected ═O, optionally protected hydroxyl, —S—, optionallyprotected thiol, —NH—, optionally protected —NH₂, optionally protected—C(O)OH, —C(O)—NH—, —C(O)—NH₂, —NH₂—C(O)—H, —NH₂—C(O)—C₀₋₄H₁₋₉,—NH₂—C(O)—O—C₀₋₄H₁₋₉, —CN, —NO₂, —N₃ or halogen.

Scheme 3. The allylic bromination at C-7 is done as in Scheme 1. R andR^(A) are as defined in Schemes 1 and 2.

Scheme 4. The addition of lithium reagent (lithium acetylide when R is—CH) to the 17-position >O═O in the presence of the bromide at C-16results in epoxide formation or in a pinacol rearrangement (not shown).Alternatively, compounds without the C₁₋₇ acetate of structure 3 can bedehydrated by mild acid catalysis to form compounds of formula 4 bytreatment of the alkene with Br₂, H₂O. R and R^(A) are as defined inSchemes 1 and 2.

Scheme 5. Sodium borohydride gives a mixture of epimers at C-7, whichmay be separated by standard methods, e.g., HPLC, TLC or columnchromatography. To obtain the pure 7α-OH compound, allylic brominationfollowed by hydrolysis is accomplished as described in Schemes 1 and 3.

Scheme 6. Formula 6 compounds are prepared by treatment of the acetate Awith lithium acetylide as in Schemes 1, 2, 3 or 4. R and R^(A) are asdefined in Schemes 1 and 2.

Scheme 7. Formula 7 compounds are prepared from the 3-acetate A withreagents described in Schemes 1 and 4. R and R^(A) are as defined inSchemes 1 and 2.

Scheme 8. Formula 8 compounds are prepared from the formula A compoundsby sodium borohydride reduction at C-17 followed by acetylation.

Scheme 9. The starting material is made using reactions described inSchemes 1 and 3. Rearrangement of the C-17 carbonyl to the C-16 positionfollowed by reduction with NaBH₄ selectively gives the C-16 β hydroxyfunction.

Scheme 10. Reduction and acetylation at C-3 and hydrolysis and oxidationat C-17 will allow formula 10a and 10b compounds to undergofunctionalization as shown in Schemes 1-9 at C-3, C-16 and C-17. The7-oxo acetate can be substituted for the formula A compound 3-acetateand functionalization at C-3, C-16 and C-17 is achieved similarly forT-oxo compounds using the reactions shown in schemes 1-9.

Treatment of 10a with LDA, followed by alkylation of the enolate allowsintroduction of side chains such as R¹⁰, which may be, e.g., C1-C20alkyl (methyl, ethyl), C1-C20 alkenyl (CH₂═CH—(CH₂)₀₋₆—), benzyl,—(CH₂)₁₋₄—O—(CH₂)₀₋₄—CH₃.

Schemes 1-9 show the introduction of the hydroxyl function at thepositions shown. Methods to convert hydroxyl to other functional groupsare accomplished essentially as described, e.g., in the references citedherein. For example, esters, of formula 1-10c compounds, such as—O—C(O)—R^(B) where R^(B) is a C₁₋₅₀ organic moiety, are prepared fromthe steroid alcohol by treatment with the appropriate acid anhydride oracid chloride (R^(B)—C(O)—Cl) to form any desired ester. Ethers, such as—O—R^(B), are prepared from alcohols by formation of the alkaline metalalkoxide (N⁺ or K⁺) followed by treatment with a primary or secondaryiodide (R^(B)—I). Thionoesters, R^(B)—C(S)—O—, are prepared by treatingthe R^(B)—C(O)—O— ester with Lawesson's reagent.

Sulfates, NaO—S(O)(O)—O—, R^(B)—O—S(O)(O)—O—, e.g.,CH₃(CH₂)₀₋₁₈—S(O)(O)—O—, are prepared by treatment of alcohols withchlorosulfonic acid followed by NaOH or alternatively by oxidation ofsulfites using KMnO₄. If the alkyl (e.g., methyl) ester is desiredalkylchloro-sulfonate (methylchloro-sulfonate) can be used. SulfitesHO—S(O)—O— and ammonium salts NH₄O—S(O)—O, or R^(B)O—S(O)—O— esters(e.g., CH₃O—S(O)—O—) are prepared by standard methods. The ammoniumsalts are prepared by treatment of alcohols with ammonia and sulfurdioxide. The esters such as alkyl, alkenyl and alkynyl esters (e.g.,methyl ester) are obtained when alcohols are treated withalkylchlorosulfite (e.g., methycholorosulfite), alkenylchlorosulfite oralkynylchlorosulfite in the presence of a suitable base such astriethylamine. Phosphoesters, R^(B)O—P(OR^(PR))(O)—O— are prepared bytreating the alcohol with diethylchlorophosphate in the presence ofNa₂CO₃. Alternatively, if the alcohol is treated with phosphoric aciddiesters in the presence of triphenylphospine (PPh₃) anddiethylazodicarboxylate (DEAD) the corresponding triesters are formedwith inversion (Mitsunobu reaction).

Phosphothioesters, R^(B)O—P(SR^(PR))(O)—O— are generated by treatment ofalcohols with the monothio analog of diethylchlorophosphate as describedfor phosphoesters yielding the phosphothioesters. Carbonates,R^(B)O—C(O)—O— are generated from the corresponding steroid alcoholusing the chloroformate (R^(B)—C(O)—Cl), e.g., C₁₋₂₀ alkyl, alkenyl oralkynyl chloroformates (e.g. CH₃(CH₂)₀₋₅—C(O)Cl). Carbamates,R^(B)—NH—C(O)—O— are made from steroid alcohols by treatment withisocyanates (R^(B)N═C═O) or NaOCN in the presence of trifluororoaceticacid. Aminoacid esters, ZNX—CHY—C(O)—O— are generated by coupling thesteroid alcohol with the acid chloride of the N-protected amino acid.

Oxidation of hydroxyl groups that are linked to the steroid nucleus isused to obtain ketones and related functionalities. For example,conversion of alcohols to ketones can be achieved using a variety ofoxidizing agents such as CrO₃ in AcOH, or pyridinium cholorchromate,pyridinium dichromate or oxalyl chloride with triethylamine (Swernoxidation). Thioketones (═S) are prepared by treating ketones withLawesson's reagent(2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide;commercially available from Aldrich). Thioacetals, —C(SR^(B))(SR^(B))—,are prepared from ketones (—C(O)—) by treatment with R^(B)—SH thiolsunder acid catalysis conditions (e.g., HCl). Phosphonoesters,RO—P(OR^(PR))(O)—, are generated by addition of the phosphorus aciddiester to ketones in the presence of KF to yield hydroxyphosphonoesters. One may optionally remove the hydroxy group using adehydration and hydrogenation sequence.

Substitution of hydroxyl groups is used to generate a number offunctionalities. For example, thiols, —SH, are prepared from alcohols byconversion of the alcohol with inversion to the bromide using PBr₃.Treatment of the bromide with thiourea followed by NaOH gives the thiol.Thioethers, R^(B)—S—, are prepared from thiols by treatment with NaOHand the required halide, e.g., alkyl halide. Alternatively, alcoholderivatives like tosylates or mesylates can be displaced by thiolateanions, R^(B)—S⁻, to yield the thioether. Thioesters, R—C(O)—S—, areprepared by treating the tosylate (mesylate) of the alcohol with thesodium salt of the thioacid.

Substitution of hydroxyl groups can be used to generate both esters,R^(B)O—C(O)—, and amides, NHR^(B)—C(O)—, linked to the steroid at carbonatoms. For amides and amines, R^(B) is —H, a protecting group or a C₁₋₅₀organic moiety. These are synthesized from the steroid bromide withinversion by displacement with NaCN. The cyanide group can be hydrolyzedto the amide or the acid. The acid is esterified or treated by standardpeptide coupling reactions with an acid-protected amino acid in thepresence of a suitable carboxyl activating agents such asdicyclohexylcarbodiimide (DCC) to form steroid —C(O)—NH—CHY—C(O)—OR,where Y is the side chain of an amino acid or a C1-C10 organic moietyand R is a protecting group (or hydrogen when deprotected).

Amines and derivatives of amines, e.g., R^(B)NH—, R^(B)—C(O)NH—,R^(B)OC(O)—NH— or R^(B)O—C(O)—CHR^(B)—NH— linked to steroid carbonatoms, are typically prepared by standard methods. For example, amines(NH₂-steroid) are generally prepared using the Hoffmann rearrangement(Br₂, NaOH) from the amide (NH₂—C(O)-steroid) or the Curtiusrearrangement (NaN₃) from the acid chloride of the steroid. The R^(B)substituent can subsequently be introduced by alkylation. Steroidalcohols can be used as starting materials under standard Mitsunobuconditions (PPh₃, DEAD) to yield N-Boc sulfonamides usingN-(t-butoxycarbonyl)-p-toluenesulfonamide. One can selectively removeeither protecting group. Treatment with trifluoroacetic acid affords thesulfonamide (R^(B)—S(O)(O)—NH-steroid). Alternatively, sodiumnapthalenide deprotects to give the N-Boc compound. Amines (NH₂-steroid)can be converted to amides (R^(B)—C(O)—NH-steroid) using acyl chlorides(R^(B)—C(O)—Cl). Treatment with ethyl chloroformate gives theN-carbamate (R^(B)O—C(O)—NH-steroid). The amine (NH₂-steroid) can bealkylated with an α-bromoester to yield the amino acid substitutedsteroid (R^(B)—O—C(O)—CHY—NH-steroid).

Where reactions such as substitutions give a product mixture, thedesired intermediate is optionally separated from other products or atleast partially enriched (e.g., enriched at least about 10-fold, usuallyat least about 50-100-fold) from other products before subsequentreactions are conducted. Substitution at steroid carbon atoms willgenerally proceed with greatest efficiency at the 3-position, which isrelatively sterically unhindered and C-17 is generally somewhat lessaccessible than the C-3 position. The relative reactivities of the C-3,C-7, C-17 and C-16 positions allows one to use their reactivities tocontrol the sequential introduction of different functional groups intothe same steroid molecule. Also, groups, such as hydroxyl at morereactive positions, C-3 or C-17, may be sequentially protected ordeprotected to allow introduction of functional groups at otherpositions, such as C-7 or C-16.

Polymers such as PEG are linked to the compounds essentially asdescribed above. For example, PEG200 or PEG300 is linked to the steroidat the 3, 7, 16, 17 or other positions by an ether linkage(PEG-O-steroid) using a PEG alkoxide (PEG-ONa), to displace the steroidbromide. Alternatively, PEG-Br can be treated with the steroid alkoxide.Polyethylene glycol esters such as those described in U.S. Pat. No.5,681,964 can also be prepared using a suitable formula 1 compound andthe methods described therein. Monosaccharides or polysaccharides andoligonucleotides are linked to steroid hydroxyl groups using knownmethods, see e.g., U.S. Pat. No. 5,627,270.

Formula 1 steroid analogs that comprise one or more ring heteroatoms aresynthesized according to the following methods.

Scheme 11. Formula 1 compounds that comprise two or three ringheteroatoms are prepared as shown in the following schemes. In thescheme, X is —CH₂—, —NH—, —O—, or —S—; R⁴⁰ is —H or —Br; R⁴¹ is anorganic moiety having about 12 carbon atoms or less, typically C1-C8optionally substituted alkyl (e.g., methyl, hydroxymethyl, ethyl,propyl, —CH(O), —CH(S)) or C2-C8 optionally substituted alkenyl having asingle double bond (e.g., vinyl) with 1, 2, 3 or more independentlyselected substituents (e.g., —OH, —COOH, —O—) and with any substituentsthat comprise a functional group generally being protected. Preparationof compound 20 from 19 is accomplished using a glycol such asHOC(CH₃)₂C(CH₃)₂OH in acid (H⁺) (B. H. Lipshutz et al., Synth. Commun.12: 267, 1982). The use of a bulky protecting group facilitatesgeneration of a double bond at the 5-6 position over the 4-5 position.

Schemes 12A-12D. Compounds of structure 12 are generated as shown in theschemes below. Most of the reactions are conducted essentially asdescribed. See e.g., W. D. Langley, Org. Syn. I, 122, 1932 (compound30); R. Ratcliffe et al., J. Org. Chem. 35: 4000, 1970 (compound 32); A.I. Meyers et al., J. Org. Chem. 39: 2787, 1974 (compound 33, 41); J. L.Isidor et al., J. Org. Chem. 38: 544, 1973 (compound 35); G. Wittig etal., Chem. Ber. 87: 1318, 1954 (compound 36); P. M. Pojer et al., Tet.Lett. 3067, 1976 (compound 38); A. Maercker, Org. React. 14: 270, 1965(compound 37); E. J. Corey et al., Tet. Lett. 3269 1975 (compound 37);R. S. Tipson, J. Org. Chem. 9: 235, 1944 (compound 39); G. W. Kabalka,J. Org. Chem. 51: 2386, 1986; B. B. Carson et al., Org. Synth. 1: 179,1941 (compound 43); H. J. Bestman et al., Justus Liebigs Ann. Chem. 693:132 1966 (compound 39); M. Miyano et al., J. Org. Chem. 37: 268, 1972(compound 51); W. H. Glaze et al., J. Org. Chem. 33: 1987, 1968(compound 52).

Compounds of structure 12 where X is NH, S and CH₂ are prepared as shownin schemes 12B, 12C and 12D respectively.

Scheme 13. The scheme and reactions shown below are used to prepare thecompound of structure 13 and related compounds that are used tointroduce oxygen, carbon, nitrogen or sulfur into the R⁷ and R⁸positions of formula 1 compounds. The reactant in the preparation ofcompound 63, 3-chloro-2-methylpropene (reg. No. 563-47-3), is availablecommercially (e.g., Aldrich, Fluka).

Compound 13 and analogs of compound 13 where CH₂, S or NH CH₂ replacesoxygen are prepared as shown in the following reactions. Conditionssuitable for conversion of compound 106 to 107 have been described (T.Hamada et al., Heterocycles 12: 647, 1979; T. Hamada et al., J. Am.Chem. Soc. 108: 140, 1986).

Conversion of the methyl ketone (—C(O)—CH₃) moiety in compounds havingthe structure

(R—C(O)—CH₃) to other functionalities is accomplished as follows. Themethyl ketone is cleaved to yield a carboxyl moiety using, e.g., Br₂ orI₂ in base, followed by treatment with acid (H₃O⁺) essentially asdescribed (S. J. Chakrabarty Oxidations in Organic Chemistry Part C, W.Trahnnousy, editor, Academic Press, NY, 1987, chapter 5; L. J. Smith etal., Org. Synth. III 302, 1953), to yield R—C(O)—OH. The carboxylic acidis converted to the acid and then reduced to the alcohol using NaBH₄.Conversion of the alcohol to the bromide is accomplished using, e.g.,Br₂ in water, essentially as described (J. S. Meck et al., Org. Synth.V, 126, 1973; A. Mckillop et al., J. Org. Chem. 34: 1172, 1969).

Compounds of structure 11 are brominated using N-bromosuccinimide toobtain steroids and analogs with bromine at the 7-position.

The 11A compounds are deprotected to yield the aldehyde compounds 12. Asshown in scheme 11, the bromine atom is ultimately found at the7-position. The bromine may be converted to a hydroxyl by reaction ofthe steroid with base (e.g., aqueous KOH), and the hydroxyl may in turnbe protected using known methods, e.g., using C₆H₅—CH₂—Br and base(KOH). The alcohol is protected and deprotected essentially usingdescribed methods, see, e.g., W. H. Hartung et al., Org. React. 7: 263,1953; E. J. Rerst et al., J. Org. Chem. 29: 3725, 1968; A. M. Felix etal., J. Org. Chem. 43: 4194, 1978; D. A. Evans et al., J. Am. Chem. Soc.101: 6789, 1979; international publication number WO 98/02450. Similarreactions are used to convert a bromine at other positions to ahydroxyl. Other substituents are linked to the steroids essentially asdescribed in schemes 1-10.

Alternative routes to introduce a functional group into the 7-positionare also suitable. For example, formula 1 compounds that have a doublebond at the 5-6 position and are unsubstituted at the 7-position areoptionally protected, e.g., hydroxyl groups are protected with acetate,and a ketone is introduced into the 7-position by oxidation with chromicacid essentially as described (U.S. Pat. No. 2,170,124). The carbonyl(═O) at 7 is reduced to a hydroxyl using mild conditions, e.g.,Al(Oi-Pr)₃, to avoid reducing the 5-6 double bond.

Selective hydrogenation of a double bond at the 16-17 position withoutreduction of a double bond at 5-6 is accomplished using H₂ and Pd. Ingeneral, ketones (═O) can be protected using a glycol, e.g., reactionwith ethylene glycol in p-toluenesulfonic acid and benzene, beforesubsequent oxidation or reduction reactions are conducted.

Various groups that may comprise the formula 1 compounds describedherein, e.g., hydroxyl groups or ketones bonded to the steroid nucleus,or substituted alkyl groups, substituted heterocycles, amino acids andpeptides, can contain one or more reactive moieties such as hydroxyl,carboxyl, amino or thiol. Intermediates used to make formula 1 compoundsmay be protected as is apparent in the art. Noncyclic and cyclicprotecting groups and corresponding cleavage reactions are described in“Protective Groups in Organic Chemistry”, Theodora W. Greene (John Wiley& Sons, Inc., New York, 1991, ISBN 0-471-62301-6) (hereafter “Greene”)and will not be detailed here. In the context of the present invention,these protecting groups are groups that can be removed from a formula 1compound without irreversibly changing the covalent bond structure oroxidation/reduction state of the remainder of the molecule. For example,the protecting group, —R^(PR), that is bonded to an —O— or —NH— groupcan be removed to form —OH or —NH₂, respectively, without affectingother covalent bonds in the molecule. At times, when desired, more thanone protecting group can be removed at a time, or they can be removedsequentially. In formula 1 compounds containing more than one protectinggroup, the protecting groups are the same or different.

Protecting groups are removed by known procedures, although it will beunderstood that the protected intermediates fall within the scope ofthis invention. The removal of the protecting group may be arduous orstraight-forward, depending upon the economics and nature of theconversions involved. In general, one will use a protecting group withexocyclic amines or with carboxyl groups during synthesis of a formula 1compound. For most therapeutic applications amine groups should bedeprotected. Protecting groups commonly are employed to protect againstcovalent modification of a sensitive group in reactions such asalkylation or acylation. Ordinarily, protecting groups are removed by,e.g. hydrolysis, elimination or aminolysis. Thus, simple functionalconsiderations will suffice to guide the selection of a reversible or anirreversible protecting group at a given locus on the formula 1compounds. Suitable protecting groups and criteria for their selectionare described in T. W. Greene and P. G. M. Wuts, Eds. “Protective Groupsin Organic Synthesis” 2nd edition, Wiley Press, at pps. 10-142, 143-174,175-223, 224-276, 277-308, 309-405 and 406-454.

Determination of whether a group is a protecting group is made in theconventional manner, e.g., as described by Kocienski, Philip J.;“Protecting Groups” (Georg Thieme Verlag Stuttgart, New York, 1994)(hereafter “Kocienski”), Section 1.1, page 2, and Greene Chapter 1,pages 1-9. In particular, a group is a protecting group if when, basedon mole ratio, 90% of that protecting group has been removed by adeprotection reaction, no more than 50%, typically 25%, more typically10%, of the deprotected product molecules have undergone changes totheir covalent bond structure or oxidation/reduction state other thanthose occasioned by the removal of the protecting group. When multipleprotecting groups of the same type are present in the molecule, the moleratios are determined when all of the groups of that type are removed.When multiple protecting groups of different types are present in themolecule, each type of protecting group is treated (and the mole ratiosare determined) independently or together with others depending onwhether the deprotection reaction conditions pertinent to one type arealso pertinent to the other types present. In one embodiment, a group isa protecting group if when, based on mole ratio determined byconventional techniques, 90% of that protecting group has been removedby a conventional deprotection reaction, no more than 50%, typically25%, more typically 10%, of the deprotected product molecules haveundergone irreversible changes to their covalent bond structure oroxidation/reduction state other than those occasioned by the removal ofthe protecting group. Irreversible changes require chemical reactions(beyond those resulting from aqueous hydrolysis, acid/baseneutralization or conventional separation, isolation or purification) torestore the covalent bond structure or oxidation/reduction state of thedeprotected formula 1 compound.

Protecting groups are also described in detail together with generalconcepts and specific strategies for their use in Kocienski, Philip J.;“Protecting Groups” (Georg Thieme Verlag Stuttgart, New York, 1994),which is incorporated by reference in its entirety herein. In particularChapter 1, Protecting Groups: An Overview, pages 1-20, Chapter 2,Hydroxyl Protecting Groups, pages 21-94, Chapter 3, Diol ProtectingGroups, pages 95-117, Chapter 4, Carboxyl Protecting Groups, pages118-154, Chapter 5, Carbonyl Protecting Groups, pages 155-184, Chapter6, Amino Protecting Groups, pages 185-243, Chapter 7, Epilog, pages244-252, and Index, pages 253-260, are incorporated with specificity inthe context of their contents. More particularly, Sections 2.3 SilylEthers, 2.4 Alkyl Ethers, 2.5 Alkoxyalkyl Ethers (Acetals), 2.6 Reviews(hydroxy and thiol protecting groups), 3.2 Acetals, 3.3 SilyleneDerivatives, 3.4 1,1,3,3-Tetraisopropyldisiloxanylidene Derivatives, 3.5Reviews (diol protecting groups), 4.2 Esters, 4.32,6,7-Trioxabicyclo[2.2.2]octanes [OBO] and Other Ortho Esters, 4.4Oxazolines, 4.5 Reviews (carboxyl protecting groups), 5.2 O,O-Acetals,5.3 S,S-Acetals, 5.4 O,S-Acetals, 5.5 Reviews (carbonyl protectinggroups), 6.2 N-Acyl Derivatives, 6.3 N-Sulfonyl Derivatives, 6.4N-Sulfenyl Derivatives, 6.5 N-Alkyl Derivatives, 6.6 N-SilylDerivatives, 6.7 Imine Derivatives, and 6.8 Reviews (amino protectinggroups), are each incorporated with specificity whereprotection/deprotection of the requisite functionalities is discussed.Further still, the tables “Index to the Principal Protecting Groups”appearing on the inside front cover and facing page, “Abbreviations” atpage xiv, and “reagents and Solvents” at page xv are each incorporatedin their entirety herein at this location.

Typical hydroxy protecting groups are described in Greene at pages14-118 and include Ethers (Methyl); Substituted Methyl Ethers(Methoxymethyl, Methylthiomethyl, t-Butylthiomethyl,(Phenyldimethylsilyl)methoxymethyl, Benzyloxymethyl,p-Methoxybenzyloxymethyl, (4-Methoxyphenoxy)methyl, Guaiacolmethyl,t-Butoxymethyl, 4-Pentenyloxymethyl, Siloxymethyl,2-Methoxyethoxymethyl, 2,2,2-Trichloroethoxymethyl,Bis(2-chloroethoxy)methyl, 2-(Trimethylsilyl)ethoxymethyl,Tetrahydropyranyl, 3-Bromotetrahydropyranyl, Tetrahydropthiopyranyl,1-Methoxycyclohexyl, 4-methoxytetrahydropyranyl,4-Methoxytetrahydrothiopyranyl, 4-MethoxytetrahydropthiopyranylS,S-Dioxido, 1-[(2-Chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl,1,4-Dioxan-2-yl, Tetrahydrofuranyl, Tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-Octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl);Substituted Ethyl Ethers (1-Ethoxyethyl, 1-(2-Chloroethoxy)ethyl,1-Methyl-1-methoxyethyl, 1-Methyl-1-benzyloxyethyl,1-Methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-Trichloroethyl,2-Trimethylsilylethyl, 2-(Phenylselenyl)ethyl, t-Butyl, Allyl,p-Chlorophenyl, p-Methoxyphenyl, 2,4-Dinitrophenyl, Benzyl); SubstitutedBenzyl Ethers (p-Methoxybenzyl, 3,4-Dimethoxybenzyl, o-Nitrobenzyl,p-Nitrobenzyl, p-Halobenzyl, 2,6-Dichlorobenzyl, p-Cyanobenzyl,p-Phenylbenzyl, 2- and 4-Picolyl, 3-Methyl-2-picolyl N-Oxido,Diphenylmethyl, p, p′-Dinitrobenzhydryl, 5-Dibenzosuberyl,Triphenylmethyl, alpha-Naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, Di(p-methoxyphenyl)phenylmethyl,Tri(p-methoxyphenyl)methyl, 4-(4′-Bromophenacyloxy)phenyldiphenylmethyl,4,4′,4″-Tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-Tris(levulinoyloxyphenyl)methyl,4,4′,4″-Tris(benzoyloxyphenyl)methyl,3-(Imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-Bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-Anthryl,9-(9-Phenyl)xanthenyl, 9-(9-Phenyl-10-oxo)anthryl,1,3-Benzodithiolan-2-yl, Benzisothiazolyl, S,S-Dioxido); Silyl Ethers(Trimethylsilyl, Triethylsilyl, Triisopropylsilyl,Dimethylisopropylsilyl, Diethylisopropylsily, Dimethylthexylsilyl,t-Butyldimethylsilyl, t-Butyldiphenylsilyl, Tribenzylsilyl,Tri-p-xylylsilyl, Triphenylsilyl, Diphenylmethylsilyl,t-Butylmethoxyphenylsilyl); Esters (Formate, Benzoylformate, Acetate,Choroacetate, Dichloroacetate, Trichloroacetate, Trifluoroacetate,Methoxyacetate, Triphenyl-methoxyacetate, Phenoxyacetate,p-Chlorophenoxyacetate, p-poly-Phenylacetate, 3-Phenylpropionate,4-Oxopentanoate (Levulinate), 4,4-(Ethylenedithio)pentanoate, Pivaloate,Adamantoate, Crotonate, 4-Methoxycrotonate, Benzoate, p-Phenylbenzoate,2,4,6-Trimethylbenzoate (Mesitoate); Carbonates (Methyl,9-Fluorenylmethyl, Ethyl, 2,2,2-Trichloroethyl, 2-(Trimethylsilyl)ethyl,2-(Phenylsulfonyl)ethyl, 2-(Triphenylphosphonio)ethyl, Isobutyl, Vinyl,Allyl, p-Nitrophenyl, Benzyl, p-Methoxybenzyl, 3,4-Dimethoxybenzyl,o-Nitrobenzyl, p-Nitrobenzyl, S-Benzyl Thiocarbonate,4-Ethoxy-1-naphthyl, Methyl Dithiocarbonate); Groups With AssistedCleavage (2-Iodobenzoate, 4-Azidobutyrate, 4-Nitro-4-methylpentanoate,o-(Dibromomethyl)benzoate, 2-Formylbenzenesulfonate,2-(Methylthiomethoxy)ethyl Carbonate, 4-(Methylthiomethoxy)butyrate,2-(Methylthiomethoxymethyl)benzoate); Miscellaneous Esters(2,6-Dichloro-4-methylphenoxyacetate,2,6-Dichloro-4-(1,1,3,3-tetramethyl-butyl)phenoxyacetate,2,4-Bis(1,1-dimethylpropyl)phenoxyacetate, Chorodiphenylacetate,Isobutyrate, Monosuccinoate, (E)-2-Methyl-2-butenoate (Tigloate),o-(Methoxycarbonyl)benzoate, p-poly-Benzoate, α-Naphthoate, Nitrate,Alkyl N,N,N′,N′-Tetramethylphosphorodiamidate, N-Phenylcarbamate,Borate, Dimethylphosphinothioyl, 2,4-Dinitro-phenylsulfenate); andSulfonates (Sulfate, Methanesulfonate (Mesylate), Benzylsulfonate,Tosylate (Tos)).

More typically hydroxy protecting groups include substituted methylethers, substituted benzyl ethers, silyl ethers, and esters includingsulfonic acid esters, still more typically, trialkylsilyl ethers,tosylates and acetates.

Typical 1,2- and 1,3-diol protecting groups are described in Greene atpages 118-142 and include Cyclic Acetals and Ketals (Methylene,Ethylidene, 1-t-Butylethylidene, 1-Phenylethylidene,(4-Methoxyphenyl)ethylidene, 2,2,2-Trichloroethylidene, Acetonide(Isopropylidene), Cyclopentylidene, Cyclohexylidene, Cycloheptylidene,Benzylidene, p-Methoxybenzylidene, 2,4-Dimethoxybenzylidene,3,4-Dimethoxybenzylidene, 2-Nitrobenzylidene); Cyclic Ortho Esters(Methoxymethylene, Ethoxymethylene, Dimethoxymethylene,1-Methoxyethylidene, 1-Ethoxyethylidine, 1,2-Dimethoxyethylidene,alpha-Methoxybenzylidene, 1-(N,N-Dimethylamino)ethylidene Derivative,alpha-(N,N-Dimethylamino)benzylidene Derivative, 2-Oxacyclopentylidene);and Silyl Derivatives (Di-t-butylsilylene Group,1,3-(1,1,3,3-Tetraiso-propyldisiloxanylidene) Derivative,Tetra-t-butoxydisiloxane-1,3-diylidene Derivative, Cyclic Carbonates,Cyclic Boronates, Ethyl Boronate, Phenyl Boronate).

More typically, 1,2- and 1,3-diol protecting groups include epoxides andacetonides.

Typical amino protecting groups are described in Greene at pages 315-385and include Carbamates (Methyl and Ethyl, 9-Fluorenylmethyl,9(2-Sulfo)fluoroenylmethyl, 9-(2,7-Dibromo)fluorenylmethyl,2,7-Di-t-buthyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]-methyl,4-Methoxy-phenacyl); Substituted Ethyl (2,2,2-Trichoroethyl,2-Trimethylsilylethyl, 2-Phenylethyl, 1-(1-Adamantyl)-1-methylethyl,1,1-Dimethyl-2-haloethyl, 1,1-Dimethyl-2,2-dibromoethyl,1,1-Dimethyl-2,2,2-trichloroethyl, 1-Methyl-1-(4-biphenylyl)ethyl,1-(3,5-Di-t-butylphenyl)-1-methylethyl, 2-(2′- and 4′-Pyridyl)ethyl,2-(N,N-Dicyclohexylcarboxamido)ethyl, t-Butyl, 1-Adamantyl, Vinyl,Allyl, 1-Isopropylallyl, Cinnamyl, 4-Nitrocinnamyl, 8-Quinolyl,N-Hydroxypiperidinyl, Alkyldithio, Benzyl, p-Methoxybenzyl,p-Nitrobenzyl, p-Bromobenzyl, p-Chorobenzyl, 2,4-Dichlorobenzyl,4-Methylsulfinylbenzyl, 9-Anthrylmethyl, Diphenylmethyl); Groups WithAssisted Cleavage (2-Methylthioethyl, 2-Methylsulfonylethyl,2-(p-Toluenesulfonyl)ethyl, [2-(1,3-Dithianyl)]methyl,4-Methylthiophenyl, 2,4-Dimethylthiophenyl, 2-Phosphonioethyl,2-Triphenylphosphonioisopropyl, 1,1-Dimethyl-2-cyanoethyl,m-Choro-p-acyloxybenzyl, p-(Dihydroxyboryl)benzyl,5-Benzisoxazolylmethyl, 2-(Trifluoromethyl)-6-chromonylmethyl); GroupsCapable of Photolytic Cleavage (m-Nitrophenyl, 3,5-Dimethoxybenzyl,o-Nitrobenzyl, 3,4-Dimethoxy-6-nitrobenzyl,Phenyl(o-nitrophenyl)methyl); Urea-Type Derivatives(Phenothiazinyl-(10)-carbonyl Derivative,N′-p-Toluenesulfonylaminocarbonyl, N′-Phenylaminothiocarbonyl);Miscellaneous Carbamates (t-Amyl, S-Benzyl Thiocarbamate, p-Cyanobenzyl,Cyclobutyl, Cyclohexyl, Cyclopentyl, Cyclopropylmethyl,p-Decyloxybenzyl, Diisopropylmethyl, 2,2-Dimethoxycarbonylvinyl,o-(N,N-Dimethyl-carboxamido)benzyl,1,1-Dimethyl-3-(N,N-dimethylcarboxamido)propyl, 1,1-Dimethylpropynyl,Di(2-pyridyl)methyl, 2-Furanylmethyl, 2-Iodoethyl, Isobornyl, Isobutyl,Isonicotinyl, p-(p′-Methoxyphenylazo)benzyl, 1-Methylcyclobutyl,1-Methylcyclohexyl, 1-Methyl-1-cyclopropylmethyl,1-Methyl-1-(3,5-dimethoxyphenyl)ethyl,1-Methyl-1-(p-phenylazophenyl)ethyl, 1-Methyl-1-phenylethyl,1-Methyl-1-(4-pyridyl)ethyl, Phenyl, p-(Phenylazo)-benzyl,2,4,6-Tri-t-butylphenyl, 4-(Trimethylammonium)benzyl,2,4,6-Trimethylbenzyl); Amides (N-Formyl, N-Acetyl, N-Choroacetyl,N-Trichoroacetyl, N-Trifluoroacetyl, N-Phenylacetyl,N-3-Phenylpropionyl, N-Picolinoyl, N-3-Pyridylcarboxamide,N-Benzoylphenylalanyl Derivative, N-Benzoyl, N-p-Phenylbenzoyl); AmidesWith Assisted Cleavage (N-o-Nitrophenylacetyl, N-o-Nitrophenoxyacetyl,N-Acetoacetyl, (N′-Dithiobenzyloxycarbonylamino)acetyl,N-3-(p-Hydroxyphenyl)propionyl, N-3-(o-Nitrophenyl)propionyl,N-2-Methyl-2-(o-nitrophenoxy)propionyl,N-2-Methyl-2-(o-phenylazophenoxy)propionyl, N-4-Chlorobutyryl,N-3-Methyl-3-nitrobutyryl, N-o-Nitrocinnamoyl, N-AcetylmethionineDerivative, N-o-Nitrobenzoyl, N-o-(Benzoyloxymethyl)benzoyl,4,5-Diphenyl-3-oxazolin-2-one); Cyclic Imide Derivatives (N-Phthalimide,N-Dithiasuccinoyl, N-2,3-Diphenylmaleoyl, N-2,5-Dimethylpyrrolyl,N-1,1,4,4-Tetramethyl-disilylazacyclopentane Adduct, 5-Substituted1,3-Dimethyl-1,3,5-triazacyclo-hexan-2-one, 5-Substituted1,3-Dibenzyl-1,3,5-triazacyclohexan-2-one, 1-Substituted3,5-Dinitro-4-pyridonyl); N-Alkyl and N-Aryl Amines (N-Methyl, N-Allyl,N-[2-(Trimethylsilyl)ethoxy]methyl, N-3-Acetoxypropyl,N-(1-Isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl), Quaternary AmmoniumSalts, N-Benzyl, N-Di(4-methoxyphenyl)methyl, N-5-Dibenzosuberyl,N-Triphenylmethyl, N-(4-Methoxyphenyl)diphenylmethyl,N-9-Phenylfluorenyl, N-2,7-Dichloro-9-fluorenylmethylene,N-Ferrocenylmethyl, N-2-Picolylamine N′-Oxide); Imine Derivatives(N-1,1-Dimethylthiomethylene, N-Benzylidene, N-p-methoxybenzylidene,N-Diphenylmethylene, N-[(2-Pyridyl)mesityl]methylene,N,(N′,N′-Dimethylaminomethylene, N,N′-Isopropylidene,N-p-Nitrobenzylidene, N-Salicylidene, N-5-Chlorosalicylidene,N-(5-Chloro-2-hydroxyphenyl)phenylmethylene, N-Cyclohexylidene); EnamineDerivative (N-(5,5-Dimethyl-3-oxo-1-cyclohexenyl)); N-Metal Derivatives(N-Borane Derivatives, N-Diphenylborinic Acid Derivative,N-[Phenyl(pentacarbonylchromium- or -tungsten)]carbenzyl, N-Copper orN-Zinc Chelate); N—N Derivatives (N-Nitro, N-Nitroso, N-Oxide); N—PDerivatives (N-Diphenylphosphinyl, N-Dimethylthiophosphinyl,N-Diphenylthiophosphinyl, N-Dialkyl Phosphoryl, N-Dibenzyl Phosphoryl,N-Diphenyl Phosphoryl); N—Si Derivatives; N—S Derivatives; N-SulfenylDerivatives (N-Benzenesulfenyl, N-o-Nitrobenzenesulfenyl,N-2,4-Dinitrobenzenesulfenyl, N-Pentachlorobenzenesulfenyl,N-2-nitro-4-methoxybenzenesulfenyl, N-Triphenylmethylsulfenyl,N-3-Nitropyridinesulfenyl); and N-Sulfonyl Derivatives(N-p-Toluenesulfonyl, N-Benzenesulfonyl,N-2,3,6-Trimethyl-4-methoxybenzenesulfonyl,N-2,4,6-Trimethoxybenzenesulfonyl,N-2,6-Dimethyl-4-methoxybenzenesulfonyl, N-Pentamethylbenzenesulfonyl,N-2,3,5,6,-Tetramethyl-4-methoxybenzenesulfonyl,N-4-methoxybenzenesulfonyl, N-2,4,6-Trimethylbenzenesulfonyl,N-2,6-Dimethoxy-4-methylbenzenesulfonyl,N-2,2,5,7,8-Pentamethylchroman-6-sulfonyl, N-methanesulfonyl,N-β-trimethylsilyethanesulfonyl, N-9-anthracenesulfonyl,N-4-(4′,8′-Dimethoxynaphthylmethyl)benzenesulfonyl, N-benzylsulfonyl,N-trifluoromethylsulfonyl, N-phenacylsulfonyl).

More typically, amino protecting groups include carbamates and amides,still more typically, N-acetyl groups.

Groups capable of biological cleavage typically include prodrugs. Alarge number of such groups are described in “Design of Prodrugs”, HansBundgaard (Elsevier, N.Y., 1985, ISBN 0-444-80675-X) (Bundgaard) andwill not be detailed here. In particular, Bundgaard, at pages 1-92,describes prodrugs and their biological cleavage reactions for a numberof functional group types. Prodrugs for carboxyl and hydroxyl groups aredetailed in Bundgaard at pages 3 to 10, for amides, imides and otherNH-acidic compounds at pages 10 to 27, amines at pages 27 to 43, andcyclic prodrugs at pages 62 to 70. These moieties are optionally bondedto the steroid at one, two or more of the variable groups that arebonded to the rings in the formula 1 compounds, e.g., one or more R¹-R⁶,R¹⁰, R¹⁵, R¹⁷ and R¹⁸.

Metabolites. Also falling within the scope of this invention are the invivo metabolites of the compounds described herein and the use of themetabolites for use in the therapeutic treatments or other methodsdescribed herein or in the cited references. This includes metabolitesor products that are novel and unobvious over the prior art as newcompounds as such and their uses. Metabolites may result for examplefrom the oxidation, reduction, hydrolysis, amidation, esterification,glycosidation and the like of the administered formula 1 compound, dueto enzymatic or chemical processes. Metabolites may be generated in vivoin a subject or they may arise ex vivo from cells or tissues, e.g., froma mammal such as a human, rodent or a primate. Accordingly, theinvention includes novel and unobvious compounds produced by a processcomprising contacting a compound of this invention with a subject or asubject's cells or tissue for a period of time sufficient to yielddetectable amounts of a metabolic product thereof. Such productstypically are identified by preparing a radiolabeled or mass labeledformula 1 compound that comprises, e.g., 1, 2, 3 or more ¹³C, ¹⁴C, ³H,²H, ¹³¹I, ³²P, ³⁵S or ⁹⁹Tc atoms bonded to the compound, andadministering it as a trace labeled compound along with the unlabeledcompound. The labeled and unlabeled compound is administered by anysuitable route (by, e.g., a buccal, sublingual, parenteral, topical ororal route) in a detectable dose (e.g. greater than about 0.1 μg/kg, orat least about 10 μg/kg or at least about 0.5 mg/kg of the labeledcompound) to a subject, e.g., an animal or mammal such as rat, mouse,guinea pig, primate, or to a human. After administration sufficient timeis allowed for metabolism to occur (typically about 30 seconds to 30hours) and conversion products are isolated from one or more of theurine, blood, plasma, feces or other suitable biological sources. Theamount of labeled formula 1 compound that is administered to a subjectwill vary with the specific activity of the labeled compound. Exemplarymetabolic conversions of formula 1 compounds include modification ofhydrogen atoms or other moieties that are bonded to, e.g., one or moreof the 1, 2, 3, 4, 6, 7, 11, 15, 16 or 17 positions. Exemplaryconversions at these one or more of positions include hydroxylation ofring atoms, e.g., ring carbon atoms, conjugation of hydroxyl groups thatare bonded to one or more of those positions with moieties such assulfate, phosphate or a monosaccharide or disaccharide such asglucuronic acid and hydrolysis of moieties such as esters or alkoxygroups.

Exemplary radiolabeled and heavy atom labeled formula 1 compoundsinclude ones that comprise 1, 2, 3 or more ¹³C, ¹⁴C, ²H, ³H, ¹³¹I, ³²Por ³⁵S atoms that are at (or bonded to), e.g., one, two, three or moreof the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18 or 19positions. In some embodiments, the molecule comprises only one or twotypes, e.g., ¹³C or ³H, of labeled atoms. Suitably labeled compoundsinclude any of the compounds disclosed herein, e.g., any formula 1compound in compound groups 1 through 54. Such labeled compounds maycomprise, e.g., a ¹³C at the 18 or 19 positions and one two or three ³Hmay be bonded to the ¹³C atom(s) or to a ring carbon(s). Other formula 1compounds may comprise one or two ²H or ³H atoms bonded to one or moreof the 1, 2, 4, 5, 6, 11 or 12 positions and optionally a ¹³C at the 18or 19 position(s).

These products or metabolites are easily isolated since they are labeled(others are isolated, e.g., by the use of antibodies capable of bindingepitopes surviving in the metabolite). The metabolite structures aredetermined in conventional fashion, e.g. by MS, GC-MS, HPLC includingreverse phase HPLC, or NMR analysis. See, e.g., H. L. J. Makin et al.,eds. Steroid Analysis 1995, Chapman & Hall, ISBN 0751401285. In general,analysis of metabolites is accomplished in the same way as conventionaldrug metabolism studies, which are known to those skilled in the art.The conversion products, especially when they are not otherwise found invivo, are useful in diagnostic assays for therapeutic dosing of theformula 1 compounds even if they possess only limited therapeuticactivity of their own.

Formulations and compositions for preparing formulations. Inventionembodiments include formulations described here and elsewhere in thisdisclosure. While it is possible for the formula 1 compound(s) to beadministered alone it is usual to present them as formulations. Theformulations, both for veterinary and for human use, comprise at leastone formula 1 compound, together with one or more excipients andoptionally one or more additional therapeutic ingredients.

This aspect of the invention includes compositions comprising one ormore pharmaceutically acceptable excipients or carriers. Thecompositions are used to prepare formulations suitable for human oranimal use. Suitable administration routes for formulations includeoral, rectal, nasal, topical (including buccal and sublingual), vaginal,rectal and parenteral (including subcutaneous, intramuscular,intravenous, intradermal, intrathecal, intraocular and epidural). Ingeneral, aqueous and non-aqueous liquid or cream formulations aredelivered by a parenteral, oral or topical route. In other embodiments,such as the invention intermittent dosing methods, the formula 1compound(s) may be present as an aqueous or a non-aqueous liquidformulation or a solid formulation suitable for administration by any ofthe routes disclosed herein, e.g., oral, topical, buccal, sublingual,parenteral, inhaled aerosol or a depot such as a subcutaneous depot oran intraperitoneal or intramuscular depot. It will be appreciated thatthe preferred route may vary with, for example, the subject'spathological condition or weight or the subject's response to therapywith a formula 1 compound or other therapy that is used or that isappropriate to the circumstances.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods known in the art ofpharmacy. Techniques, excipients and formulations generally are foundin, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa. 1985, 17^(th) edition, Nema et al., PDA J. Pharm. Sci. Tech.1997 51:166-171, G. Cole, et al., editors, Pharmaceutical CoatingTechnology, 1995, Taylor & Francis, ISBN 0 136628915, H. A. Lieberman,et al., editors, Pharmaceutical Dosage Forms, 1992 2^(nd) revisededition, volumes 1 and 2, Marcel Dekker, ISBN 0824793870, J. T.Carstensen. Pharmaceutical Preformulation, 1998, pages 1-306, TechnomicPublishing Co. ISBN 1566766907. Exemplary excipients for formulationsinclude emulsifying wax, propyl gallate, citric acid, lactic acid,polysorbate 80, sodium chloride, isopropyl palmitate, glycerin, whitepetrolatum and other excipients disclosed herein.

Methods to make invention formulations include the step of bringing intoassociation or contacting a formula 1 compound(s) with one or moreexcipient, such as one described herein or in the cited references. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the formula 1 compound(s) with liquidexcipients or finely divided solid excipients or both, and then, ifappropriate, shaping the product.

Formulations suitable for oral administration are prepared as discreteunits such as capsules, cachets or tablets each containing apredetermined amount of the formula 1 compound(s); as a powder orgranules; as solution or a suspension in an aqueous liquid or anon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The formula 1 compound(s) may also bepresented as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the formula 1 compound(s) in a free-flowing formsuch as a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered or granulated formula 1 compound and one or more excipients,which are optionally moistened, with an inert liquid diluent orexcipient. The tablets may optionally be coated or scored and optionallyare formulated so as to provide slow or controlled release of theformula 1 compound(s) therefrom. An exemplary tablet or caplet (acapsule shaped tablet) formulation suitable for buccal or sublingualdelivery of a formula 1 compound to a subject's tissues comprises about25 or 50 mg of a formula 1 compound such as BrEA hemihydrate comprisingper 25 mg of the formula 1 compound about 6.2 mg povidone, about 0.62 mgmagnesium stearate, about 45 mg mannitol and about 48 mg of compressiblesucrose.

For infections of the eye or other external tissues e.g., the mouth orskin, the formulations are typically applied as a topical ointment orcream containing the formula 1 compound(s) in an amount of, for example,about 0.075 to about 20% w/w (including formula 1 compound(s) in a rangebetween about 0.1% and 20% in increments of 0.1% w/w such as about 0.6%w/w, about 0.7% w/w, about 1% w/w, about 1.5% w/w, about 2% w/w, about2.5 w/w, about 3% w/w, about 5% w/w, about 7% w/w, about 10% w/w etc.),including about 0.2 to 15% w/w and about 0.5 to 10% w/w. When formulatedin an ointment, the formula 1 compound(s) may be employed with either aparaffinic or a water-miscible ointment base. Alternatively, they may beformulated in a cream with an oil-in-water cream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, butane 1,4-diol, mannitol, sorbitol, glycerol and apolyethylene glycol (including, e.g., PEG 300 and PEG 400) and mixturesthereof. The topical formulations may include a compound that enhancesabsorption or penetration of the formula 1 compound(s) through the skinor other affected areas. Examples of such dermal penetration enhancersinclude dimethyl sulphoxide and related analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known excipients in a known manner. While the phase may comprise anemulsifier (otherwise known as an emulgent), it desirably comprises amixture of at least one emulsifier with a fat or an oil or with both afat and an oil. A hydrophilic emulsifier may be included together with alipophilic emulsifier, which acts as a stabilizer. Some embodimentsinclude both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationsinclude Tween60™, Span80™, cetostearyl alcohol, benzyl alcohol, myristylalcohol, glyceryl mono-stearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. Creams are generally anon-greasy, non-staining and washable products with suitable consistencyto avoid leakage from tubes or other containers. Straight or branchedchain, mono- or dibasic alkyl esters such as di-isoadipate, isocetylstearate, propylene glycol diester of coconut fatty acids, isopropylmyristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used. These may be used alone or in combinationdepending on the properties required. Alternatively, high melting pointlipids such as white soft paraffin and/or liquid paraffin or othermineral oils are used.

Formulations suitable for topical administration to the eye include eyedrops wherein the formula 1 compound(s) is dissolved or suspended in asuitable excipient(s), including an aqueous solvent for a formula 1compound(s) that comprise at least about 0.5, one, two or more chargesat pH values near neutrality, e.g., about pH 6-8. The formula 1compound(s) is typically present in such formulations in a concentrationof about 0.5-20% w/w, about 1-10% w/w or about 2-5% w/w.

Formulations suitable for topical administration to oral mucosa includelozenges or tablets comprising the formula 1 compound(s) in a flavoredbasis or a monosaccharide or disaccharide such as sucrose, lactose orglucose and acacia or tragacanth; pastilles comprising the formula 1compound(s) in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the formula 1 compound(s) in asuitable liquid excipient(s). In some embodiments, the lozenges ortablets optionally comprise the property of rapid dissolution ordisintegration, e.g., disintegration within about 15 seconds to about 2minutes, while in others, the lozenges or tablets comprise the propertyof slower dissolution or disintegration, e.g., disintegration withinabout 2 minutes to about 10 minutes or more.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the formula 1 compound(s) such excipients asare known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats, salts (e.g., NaCl, potassium or sodium carbonateor bicarbonate or potassium or sodium phosphates) and solutes whichrender the formulation isotonic with the blood of the intended subject;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. In general, the formula 1compound that is present in liquid compositions or formulations iscompletely dissolved in aqueous or non-aqueous excipients. However, insome embodiments, e.g., transient compositions or some formulations, theformula 1 compound is partially dissolved while the remaining portion ispresent as a solid, which can be a suspension or a colloid.

Exemplary formulations suitable for parenteral delivery of formula 1compounds to subjects such as humans or animals typically comprise one,two, three or more excipients. Exemplary embodiments include (1) anytwo, three or four of propylene glycol, PEG200, PEG300, ethanol andbenzyl benzoate and (2) any two, three or four of propylene glycol,PEG100, PEG200, PEG300, PEG400 and benzyl benzoate. Another exemplaryformulation suitable for parenteral use include an aqueous

BrEA hemihydrate suspension comprising about 50-120 mg/mL of BrEAhemihydrate that has an average particle size of 20 μm or less, about0.05-0.2% w/v carboxymethylcellulose sodium, about 1-3% w/v polysorbate80, about 0.75-0.85% w/v NaCl, about 0.023% w/v dibasic sodiumphosphate, about 0.101% w/v monobasic sodium phosphate, about 0-0.5%ethanol v/v, pH 6.5+/−0.4 and optionally about 0.1-0.3% w/v of apreservative such as methylparaben.

Exemplary compositions and formulations generally comprise about0.01-10% of a formula 1 compound, usually about 1-5%, and about 0.01-3%water, typically about 0.05-3%, usually about 0.1-1%. The formulationsinclude unit or multi-unit dosages suitable for parenteraladministration once or twice per day or once per 2-3 days. Unit dosagescomprise about 3-1000 mg of formula 1 compound per unit dose, typicallyabout 5-500 mg, usually about 10-200 mg. For treating retroviruses suchas HIV in humans, a unit dose usually comprises about 10-250 mg of BrEAhemihydrate, usually about 100-200 mg, in a volume of about 1-6 mL,usually about 2-4 mL.

Formulations, or compositions disclosed herein for use to makeformulations suitable for administration by the routes disclosed hereinoptionally comprise an average particle size in the range of about 0.01to about 500 microns, about 0.1 to about 100 microns or about 0.5 toabout 75 microns. Average particle sizes include a range between 0.01and 500 microns in 0.05 micron or in 0.1 micron or other increments,e.g., an average particle size of about 0.05, 0.1, 0.5, 1, 1.5, 2.0,2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 50, 60, 75, 85, 100, 120, etc. microns). When formula 1 compounds orcompositions that comprise a formula 1 compound are used asintermediates to make a formulation, they may comprise one, two, threeor more of these average particle sizes, or size ranges. In preparingany of the compositions or formulations that are disclosed herein andthat comprise a formula 1 compound (and optionally one or moreexcipients), one may optionally mill, sieve or otherwise granulate thecompound or composition to obtain a desired particle size, e.g., asdescribed above.

Milling may occur before or after the formula 1 compound is contactedwith one or more excipients. For example, one may mill a formula 1compound such as 16α-bromoepiandrosterone hemihydrate, to obtain anaverage particle size (or diameter) of about 0.05-50 μM or about 0.5-10μM (e.g., about 0.04, 0.1, 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 20, 40, 60,80, 100 or 120 μM average particle size or diameter) before contactingthe milled formula 1 compound with a liquid or solid excipient. In somecases the formula 1 compound is milled or sieved to obtain an averageparticle size of about 5 μm or about 10 μm before it is contacted with asolid or liquid excipient(s) to obtain a solution or suspension or apowder suitable for making a tablet, capsule or other dosage form asdescribed herein or in the cited references.

As used herein, reference to an average particle size or an averageparticle diameter means that the material, e.g., a formula 1compound(s), an excipient(s) or a composition that comprises both, isground, milled, sieved or otherwise treated so as to comprise thespecified average size. It is to be understood that some particles maybe larger or smaller, but the composition or the formula 1 compound(s)will comprise a significant proportion of the material with thespecified size or within an acceptable range of the specified size.Micronization methods include milling by ball mills, pin mills, jetmills (e.g., fluid energy jet mills) and grinding, sieving andprecipitation of a compound(s) from a solution, see, e.g., U.S. Pat.Nos. 4,919,341, 5,202,129, 5,271,944, 5,424,077 and 5,455,049. Averageparticle size is determined by, e.g., transmission electron microscopy,scanning electron microscopy, light microscopy, X-ray diffractometry andlight scattering methods or Coulter counter analysis.

Thus, the formula 1 compounds may comprise a powder that consists ofone, two or more of these average particle sizes and the powder may becontacted with a solid excipient(s), suitably mixed and optionallycompressed or formed into a desired shape. Alternatively, such a formula1 compound(s) is contacted with a liquid excipient(s) to prepare aliquid formulation or a liquid composition that is incorporated into asolid formulation. Suitable micronized formulations thus include aqueousor oily solutions or suspensions of the formula 1 compound(s).

Formulations suitable for aerosol administration typically will comprisea fine powder, e.g., having an average particle size of about 0.1 toabout 20 microns or any one, two or more of the average particle sizeswithin this range that are described above. The powder is typicallydelivered by rapid inhalation through the nasal passage or by inhalationthrough the mouth so as to reach the bronchioles or alveolar sacs of thelungs.

Formulations suitable for aerosol, dry powder or tablet administrationmay be prepared according to conventional methods and may be deliveredwith other therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis of viral or other infections as describedherein. Such formulations may be administered, e.g., orally,parenterally (e.g., intravenous, intramuscular, subcutaneous,intradermal, intrathecal), topically, sublingually or by a buccal orsublingual route.

Micronized formula 1 compound is useful, e.g., to facilitate mixing,dissolution or uniform suspension of the formula 1 compound in one ormore liquid or solid excipients, e.g., a PEG such as PEG 300 or PEG 400,or propylene glycol or benzyl benzoate, a complexing agent, such as acyclodextrin (e.g., an α-, β- or γ-cyclodextrin such ashydroxypropyl-β-cyclodextrin). Micronized formula 1 compound is alsouseful to facilitate uniformly distributing drug substance when themicronized compound is contacted with one or more solid excipients(e.g., a filler, a binder, a disintegrant, complexing agent (e.g., acyclodextrin such as hydroxypropyl-β-cyclodextrin), a preservative, abuffer or a lubricant).

In related embodiments, suitable compositions or formulations comprise aformula 1 compound that is present in two or more physical forms. Forexample, a liquid composition or formulation may comprise a formula 1compound that is present in solution and as undissolved particles, whichmay be milled as described herein. Alternatively, a solid composition orformulation may comprise a formula 1 compound that is present as anamorphous form and as a crystal or in an encapsulated granule. Suchencapsulated granules may comprise a slow release type formulation andthe formula 1 compound that is present may be in one or more physicalforms, e.g., liquids or solids as described herein, but usually as asolid in tablets or other solid formulations.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets as described above.Unit dosage formulations are those containing a daily dose or unit dailysub-dose, as recited herein, or an appropriate fraction thereof, of theformula 1 compound(s).

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents or excipients conventional in the art having regard to the typeof formulation in question, for example those suitable for oraladministration may include flavoring agents.

Formulations made from or comprising a formula 1 compound such as BrEAhemihydrate are optionally stored under conditions that limit the amountof light or water that reaches the formulation, e.g., in a sealedcontainer that holds a formulation or unit dosage form and optionallycontains silica gel or activated carbon. Water permeationcharacteristics of containers have been described, e.g.,Containers—Permeation, Chapter, USP 23, 1995, U.S. PharmacopeialConvention, Inc., Rockville, Md., p. 1787. Storage of BrEA hemihydrateor formulations that contain it is typically at about 4-30° C.

The invention further provides veterinary compositions comprising atleast one formula 1 compound together with a veterinary excipient(s)therefor. Veterinary excipients are materials useful for the purpose ofadministering the composition and may be solid, liquid or gaseousmaterials that are otherwise inert or acceptable in the veterinary artand are compatible with the formula 1 compound(s). These veterinarycompositions may be administered orally, parenterally or by any otherdesired route.

Invention formulations include controlled release pharmaceuticalformulations containing a formula 1 compound(s) (“controlled releaseformulations”, “slow release formulations” or the like) in which therelease of the formula 1 compound(s) is controlled or regulated to allowless frequency dosing or to improve the pharmacokinetic or toxicityprofile of a given formula 1 compound(s). Polymers and other materialsthat are suitable to prepare controlled release formulations thatcomprise a formula 1 compound have been described, e.g., U.S. Pat. Nos.4,652,443, 4,800,085, 4,808,416, 5,013,727, 5,188,840.

Thus, microcapsules, granules or other shaped forms may comprise aformula 1 compound and a slow release polymer or polymer matrix thatcomprises or consists of one or more of ethylene dimethacrylate,diethylene glycol dimethacrylate, diethylene glycol diacrylate,triethylene glycol dimethacrylate, triethylene glycol diacrylate,tetrathylene glycol dimethacrylate, tetraethylene glycol diacrylate,polyethylene glycol dimethacrylate, polyethylene glycol diacrylate,diethylaminoethyl dimethacrylate, glycidyl methacrylate, epoxy acrylate,glycidyl acrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate,hydroxypropyl methacrylate, hydroxypropyl acrylate, hydroxybutylmethacrylate, hydroxybutyl acrylate, hydroxyhexyl methacrylate,hydroxyhexyl acrylate, butanediol dimethacrylate, butanediol diacrylate,propanediol dimethacrylate, propanediol diacrylate, pentanedioldimethacrylate, pentanediol diacrylate, hexanediol dimethacrylate,hexanediol diacrylate, neopentyl glycol dimethacrylate, neopentyl glycoldiacrylate, trimethylopropane triacrylate, trimethylolpropanetrimethacrylate, trimethyloethane triacrylate, trimethylolethanetrimethacrylate, polypropyleneglycol diacrylate, and polypropyleneglycol dimethacrylate.

An effective dose of formula 1 compound(s) depends in part on one ormore of the nature of the condition being treated, toxicity, whether theformula 1 compound(s) is being used prophylactically (generally lowerdoses) or against an active infection or condition, the method ofdelivery, and the formulation. For use in humans, the effective dosagewill typically be determined by the clinician using conventional doseescalation studies. In other subjects, the dose is obtained by similardose escalation analyses. For humans, an effective dose can be expectedto be from about 0.05 to about 50 mg/kg body weight per day, e.g., about2 mg/kg/day, about 4 mg/kg/day, about 6 mg/kg/day, or about 8 mg/kg/day.For example, for topical delivery the daily candidate dose for an adulthuman of approximately 70 kg body weight will range from about 1 mg toabout 500 mg, generally between about 5 mg and about 40 mg, and may takethe form of single or multiple doses or administration sites. Fornon-human subjects, e.g., mammals such as rodents or primates, theeffective daily dosage may comprise about 0.05 mg/kg/day to about 300mg/kg/day, including about 1 mg/kg/day to about 100 mg/kg/day. As usedherein, an “effective dosage” or an “effective amount” of a formula 1compound(s) is one that is sufficient to result in, e.g., a detectablechange in a symptom or an immune parameter such as one described herein.

An effective dosage (or daily dosage) may be administered to a subjectover a period of time, e.g., at least about 1-14 days before a symptomchange or an immune parameter detectably changes. Effective dosages mayinclude any of the dosages as described herein.

Embodiments include formulations that comprise a liposome or lipidcomplex that comprises a formula 1 compound(s), e.g., BrEA or an ester,carbamate, carbonate, amino acid or peptide thereof. Such formulationsare prepared according to known methods, e.g., U.S. Pat. Nos. 4,427,649,5,043,165, 5,714,163, 5,744,158, 5,783,211, 5,795,589, 5,795,987,5,798,348, 5,811,118, 5,820,848, 5,834,016 and 5,882,678. The liposomesoptionally contain an additional therapeutic agent(s), e.g.,amphotericin B, cis-platin, adriamycin, a protease inhibitor, anucleoside or a nucleotide analog, such as one of those mentionedherein. Formulations that comprise liposomes can be delivered to asubject by any standard route, e.g., oral, aerosol or parenteral (e.g.,s.c., i.v. or i.m.).

Liposome formulations can be used to enhance delivery of the formula 1compound(s) to certain cell types such as tumor cells (see e.g., U.S.Pat. No. 5,714,163) or to cells of the reticuloendothelial system(“RES”). The RES includes macrophages, mononuclear phagocytic cells,Kupfer cells, cells lining the sinusoids of the spleen, lymph nodes, andbone marrow, and the fibroblastic reticular cells of hematopoietictissues. In general, RES cells are phagocytic and they are targets fortargeted delivery of a formula 1 compound(s) in vitro or in vivo usingliposomes, or other compositions or formulations. Thus, one can deliverformula 1 compound to a neoplasm that is derived fromreticuloendothelial tissue (reticuloendothelioma). The liposomes mayoptionally comprise a peptide from an infectious agent such as a malariaparasite, a virus or a tumor associated antigen. The peptides mayfacilitate the generation of a MHC class II and B cell response.

Invention embodiments include the product made by a process ofcombining, mixing or otherwise contacting a formula 1 compound such asBrEA hemihydrate and one, two or more excipients. Such products areproduced by routine methods of contacting the ingredients. Such productsoptionally contain a diluent, a disintegrant, a lubricant, a binder, orother excipients described herein or in references cited herein.

Other embodiments include compositions that transiently occur when amethod step or operation is performed. For example, when a formula 1compound such as BrEA, containing less than about 3% water is contactedwith an excipient, e.g., a PEG, an alcohol, propylene glycol or benzylbenzoate, the composition before addition of one ingredient with anotheris a non-homogenous mixture. As the ingredients are contacted, themixture's homogeneity increases and the proportion of ingredientsrelative to each other approaches a desired value. Thus, inventioncompositions, which contain less than about 3% water can comprise about0.0001-99% of a formula 1 compound such as BrEA and one or moreexcipients. These transient compositions are intermediates thatnecessarily arise when one makes an invention composition or formulationand they are included in invention embodiments to the extent that theyare useful in the disclosed methods or that they are patentable.

When a formula 1 compound and an excipient(s) is contacted or mixed, thefinal composition may comprise a homogenous mixture or it may comprise amixture that is not homogenous for one or more of the compounds that arepresent in the composition. Compositions and formulations that areeither homogenous or non-homogenous are included in the scope of theinvention. Non-homogenous compositions can be used to make controlledrelease formulations.

Invention embodiments include compositions and formulations thatcomprise less than about 3% water, a formula 1 compound and a compoundthat is not generally considered suitable for human use but is useful tomake an invention formulation for veterinary use. Veterinaryformulations are compositions useful for the purpose of administeringinvention compositions to primates, cats, dogs, horses, cows, rabbitsand other subjects and may contain excipients acceptable in theveterinary art and are compatible with formula 1 compounds such as BrEA.These veterinary compositions may not always be suitable for human usebecause they contain an excipient that is not suitable for human use,e.g., an alcohol other than ethanol such as methanol, propanol orbutanol. Typically such excipients will be present at relatively lowlevels, e.g., about 1-30%, usually about 1-5%.

Invention embodiments include compositions and formulations, e.g., unitdosage forms and sterile solutions, that comprise (1) about 1-100 mg/mLof a formula 1 compound(s), about 57.5% propylene glycol, about 25%PEG300, about 12.5% ethanol and about 5% benzyl benzoate; (2) about 1-60mg/mL of a formula 1 compound(s), about 70% propylene glycol, about 25%PEG300 and about 5% benzyl benzoate; (3) about 1-60 mg/mL of a formula 1compound(s), about 25% PEG300, about 35% propylene glycol, about 35%mannitol and about 5% benzyl benzoate; (4) about 1-60 mg/mL of a formula1 compound(s), about 57.5% propylene glycol, a mixture comprising about25% PEG300 and PEG200 (e.g., PEG300:PEG200 in a ratio of about 1:10 toabout 10:1), about 12.5% ethanol and about 5% benzyl benzoate; (5) about1-60 mg/mL of a formula 1 compound(s), about 75% propylene glycol, amixture comprising about 25% PEG300 and PEG200 (e.g., a PEG300:PEG200 ina ratio of about 1:10 to about 10:1) and about 5% benzyl benzoate; (6)about 1-60 mg/mL of a formula 1 compound(s), about 25% PEG300 and PEG200(e.g., PEG300:PEG200 in a ratio of about 1:10 to about 10:1), about 35%propylene glycol, about 35% mannitol and about 5% benzyl benzoate; (7)any of formulations (1) through (6) where the formula 1 compound(s) isabout 40-55 mg/mL; (8) any of formulations (1) through (6) where theformula 1 compound(s) is about 30-100 mg/mL; (9) any of formulations (1)through (8) where 1, 2, 3 or 4 formula 1 compounds are present; (10) anyof formulations (1) through (8) where 1 or 2 formula 1 compounds arepresent; (11) any of formulations (1) through (8) where 1 formula 1compound is present; (12) any of formulations (1) through (11) where theformula 1 compound comprises independently at 1, 2 or 3 of any of thevariable groups that are bonded to the formula 1 compounds, e.g., R¹-R⁶,R¹⁰, R¹⁵, R¹⁷ or R¹⁸, an independently selected ester, thioester,carbonate, carbamate, amino acid or peptide of 1 or 2 independentlyselected formula 1 compounds; (13) any of formulations (1) through (12)where the formula 1 compound comprises or is BrEA or BrEA hemihydrate;(14) any of formulations (1) through (13) where the formula 1 compoundcomprises or is an ester, a sulfate ester, a monosaccharide conjugate orphosphoester of BrEA.

Exemplary embodiments include liquid formulations that comprise aformula 1 compound, one, two, three, four or more excipients and lessthan about 3% v/v water, wherein the formulation is optionally disposedin containers that exclude water. These excipients are optionallyselected from those disclosed herein. Such formulations optionallycomprise less than about 2% v/v water, less than about 1% v/v water,less than about 0.5% v/v water, less than about 0.2% v/v water or lessthan about 0.1% v/v water. Such formulations are suitable for use inmethods to modulate an immune response or cellular response in a subjectin need thereof comprising administering to the subject, or deliveringto the subject's tissues, an effective amount of a compound offormula 1. The subject in need thereof would have, or be subject to, acondition such as one disclosed herein as amenable to treatment,prevention, amelioration using a formula 1 compound, which is optionallycombined with the use of another therapeutic treatment or agent asdisclosed herein or in the cited references. These formulations aresuitable for use in any of the dosing methods or protocols disclosedherein, including the intermittent dosing protocols disclosed herein.

Buccal formulations. Formula 1 compounds may be administered to subjectsby buccal or sublingual dosing. The buccal area generally refers to thesubject's mouth and pharynx, and the buccal mucosa includes the mucosaof the mouth and pharynx. The sublingual area refers generally to themucosa below and adjacent to the tongue. Formulations suitable forbuccal or sublingual administration typically comprise about 1-100 mg offormula 1 compound per unit dose, often about 2-60 mg. Buccal orsublingual formulations may comprise a tablet that contains about 1, 5,10, 15, 20, 25, 30, 35, 40, 50 or 60 mg of a formula 1 compound. Solidand liquid buccal or sublingual formulations optionally include one,two, three or more excipients such as fillers, binders, lubricants,antioxidants, preservatives, flavoring agents or disintegrants, e.g.,lactose, sucrose, mannitol, Tween-80, magnesium stearate, butylatedhydroxyanisole, butylated hydroxytoluene, cyclodextrins (e.g.,α-cyclodextrins, β-cyclodextrins, γ-cyclodextrins,hydroxypropyl-β-cyclodextrin), carbomers, hydrolyzed polyvinylalcohol,polyethylene oxide, polyacrylates, hydroxypropylmethylcellulose,hydroxypropylcellulose, and combinations thereof. Such formulations maybe a unit solid such as a tablet, or a powder or liquid. Buccal tabletsmay comprise a concave surface for contacting the buccal mucosa andadhering to it. A buccal or sublingual dosage may comprise a compressedtablet of a substantially uniform mixture of a bioerodible polymericcarrier, which on sustained contact with the oral mucosa, substantiallyor completely erodes within a predetermined period in the range of about10 minutes to about 24 hours. In some embodiments, the formula 1compound is administered by a method for administering the compound tothe subject, e.g., to a mammal or a human, comprising affixing a unitdosage or tablet to the subject's buccal mucosa in a region at or nearthe upper gum between the first bicuspid on the left and the firstbicuspid on the right (or an alternative location for the dosage unit isthe inner lip area opposing the this upper gum area) and optionallyallowing the tablet to remain in place until erosion thereof is completeor nearly complete. Exemplary excipients may comprise a combination ofpolyethylene oxide and a carbomer, e.g., wherein the polyethylene oxideand the carbomer are in an approximately 1:5 to 5:1 ratio by weight.

Tablets or unit dosages for buccal or sublingual delivery may be about 5mm in diameter and 2 mm in height, so that the unit dosage occupiesabout 40 mm³. Such dosages will typically weigh less than about 100 mg(e.g., about 5 to 60 mg), with a contact surface area of about 10-30mm², e.g., about 15-20 mm². Such dosages will generally be about 4-10 mmin diameter and about 1-3 mm in height. When a polymer excipient isused, it optionally comprises a polymer having sufficient tack to ensurethat the dosage unit adheres to the buccal mucosa for a sufficient timeperiod, e.g., the time period during which drug is to be delivered tothe buccal mucosa. The polymeric excipient is gradually “bioerodible,”and it hydrolyzes, dissolves, erodes or disintegrates (collectively“erodes”) at a predetermined rate upon contact with water or saliva. Thepolymeric carrier is generally sticky when moist, but not when dry, forconvenience in handling. The average molecular weight of the polymer maybe about 400 to 1,000,000, or about 1,000 to 100,000. Higher themolecular weight polymers generally erode more slowly.

For these buccal and sublingual dosages, a pharmaceutically acceptablepolymer(s) can be used. Such polymers will provide a suitable degree ofadhesion and the desired drug release profile, and are generallycompatible with the drug to be administered and any other componentsthat may be present in the buccal dosage unit. The polymeric carriersoptionally comprise hydrophilic (water-soluble and water-swellable)polymers that adhere to the wet surface of the buccal mucosa. Examplesof polymeric carriers that are useful herein include acrylic acidpolymers and co, e.g., those known as “carbomers” (Carbopol™, which maybe obtained from B.F. Goodrich, is one such polymer). Other suitablepolymers include hydrolyzed polyvinylalcohol; polyethylene oxides (e.g.,Sentry Polyox™ water soluble resins, available from Union Carbide);polyacrylates (e.g., Gantrez™, which may be obtained from GAF); vinylpolymers and copolymers; polyvinylpyrrolidone; dextran; guar gum;pectins; starches; and cellulosic polymers such as hydroxypropylmethylcellulose, (e.g., Methocel™, which may be obtained from the DowChemical Company), hydroxypropyl cellulose (e.g., Klucel™, which may beobtained from Dow), hydroxypropyl cellulose ethers (see, e.g., U.S. Pat.No. 4,704,285 to Alderman), hydroxyethyl cellulose, carboxymethylcellulose, sodium carboxymethyl cellulose, methyl cellulose, ethylcellulose, cellulose acetate phthalate, cellulose acetate butyrate, andthe like. The carrier may also comprise two or more suitable polymers incombination, for example, a carbomer combined in an approximately 1:5 to5:1 ratio, by weight, with a polyethylene oxide.

Buccal dosages may contain only the formula 1 compound and thepolymer(s). However, it may be desirable in some cases to include one ormore additional excipients. For example, a lubricant may be included tofacilitate the process of manufacturing the dosage units; lubricants mayalso optimize erosion rate and drug flux. If a lubricant is present, itmay optionally represent about 0.01 wt. % to about 2 wt. %, or about0.01 wt. % to 0.5 wt. %, of the dosage unit. Suitable lubricantsinclude, but are not limited to, magnesium stearate, calcium stearate,stearic acid, sodium stearylfumarate, talc, hydrogenated vegetable oilsand polyethylene glycol. However, modulating the particle size of thecomponents in the dosage unit and/or the density of the unit can providea similar effect, e.g., improved man ufacturability, and optimization oferosion rate and drug flux without addition of a lubricant.

Other excipients are also optionally incorporated into buccal unitdosages. Such additional optional excipients include, one or moredisintegrants, diluents, binders, enhancers, or the like. Examples ofdisintegrants that may be used include, but are not limited to,cross-linked polyvinylpyrrolidones, such as crospovidone (e.g.,Polyplasdone™ XL, which may be obtained from GAF), cross-linkedcarboxylic methylcelluloses, such as croscarmelose (e.g., Ac-di-sol™,which may be obtained from FMC), alginic acid, and sodium carboxymethylstarches (e.g., Explotab™, which may be obtained from Edward Medell Co.,Inc.), methylcellulose, agar bentonite and alginic acid. Suitablediluents are those which are generally useful in pharmaceuticalformulations prepared using compression techniques, e.g., dicalciumphosphate dihydrate (e.g., Di-Tab™, which may be obtained fromStauffer), sugars that have been processed by cocrystallization withdextrin (e.g., co-crystallized sucrose and dextrin such as Di-Pak™,which may be obtained from Amstar), lactone, calcium phosphate,cellulose, kaolin, mannitol, sodium chloride, dry starch, powdered sugarand the like. Binders, if used, are those that enhance adhesion.Examples of such binders include, but are not limited to, starch,gelatin and sugars such as sucrose, dextrose, molasses, and lactose.Permeation enhancers may also be present in the novel dosage units inorder to increase the rate at which the active agent passes through thebuccal mucosa. Examples of permeation enhancers include, but are notlimited to, polyethylene glycol monolaurate (“PEGML”), glycerolmonolaurate, lecithin, the 1-substituted azacycloheptan-2-ones,particularly 1-n-dodecylcyclaza-cycloheptan-2-one (available under thetrademark Azone™ from Nelson Research & Development Co., Irvine,Calif.), lower alkanols (e.g., ethanol), SEPA™ (available from MacrochemCo., Lexington, Mass.), cholic acid, taurocholic acid, bile salt typeenhancers, and surfactants such as Tergitol™, Nonoxynol-9™ andTWEEN-80™.

Flavorings are optionally included in buccal or sublingual formulations.Any suitable flavoring may be used, e.g., one or more of mannitol,sucrose, glucose, lactose, lemon, lemon lime, orange, menthol orartificial sweeteners such as aspartame, saccharin sodium, dipotassiumglycyrrhizinate, stevia and thaumatin. Some sweeteners such as sucrosemay also aid in dissolution or erosion of solid formulations. Coloringagents may also be added, e.g., any of the water soluble FD&C dyes ormixtures thereof, e.g., one or more of FD&C Yellow No. 5, FD&C RED No.2, FD&C Blue No. 2, etc., food lakes or red iron oxide. In addition suchformulations dosages may be formulated with one or more preservatives orbacteriostatic agents, e.g., methyl hydroxybenzoate, propylhydroxybenzoate, chlorocresol, benzalkonium chloride, or the like.

Other embodiments include solid buccal or sublingual formulationscomprising (i) a formula 1 compound and (ii) erythritol, (iii)crystalline cellulose and (iv) a disintegrant, e.g., crospovidone. Theseformulations are capable of buccal disintegration or dissolution and mayfurther comprise mannitol. These formulations may dissolve completely insolely saliva within about 1-10 minutes of administration to a subject.The erythritol is optionally contained in a proportion of about 5-90parts by weight, based on 100 parts by weight of the solid buccalformulation. The crystalline cellulose is optionally contained in aproportion of about 3-50 parts by weight, based on 100 parts by weightof the formulation. The disintegrant is optionally contained in aproportion of 1-10 parts by weight. In any of the solid buccal orsublingual formulations the ingredients are generally uniformly mixed,although non-uniform mixtures may be used. An exemplary formulationcomprises a solid capable of buccal disintegration or dissolution, whichcomprises (i) about 0.3-50 parts by weight of a formula 1 compound, (ii)about 50-80 parts by weight of erythritol, (iii) about 5-20 parts byweight of crystalline cellulose and (iv) about 3-7 parts by weight of adisintegrant, which optionally is one or more of crospovidone,croscarmellose, croscarmellose sodium, carmellose calcium,carboxymethylstarch sodium, low substituted hydroxypropyl cellulose orcorn starch. Examples of the crystalline cellulose include products ofvarious grade such as CEOLUS KG801, avicel PH101, avicel PH102, avicelPH301, avicel PH302, avicel RC-591 (crystalline cellulose carmellosesodium) and so on. One crystalline cellulose may be used or two or morespecies may be used in combination. The disintegrant, e.g.,crospovidone, may be used singly or in combination with otherdisintegrants. Crospovidone includes any cross-linked1-ethenyl-2-pyrrolidinone homopolymer, and may comprise a polymer ofmolecular weight of 1,000,000 or more. Examples of commerciallyavailable crospovidone include Cross-linked povidone, Kollidon CL,Polyplasdone XL, Polyplasdone XL-10, INF-10 (manufactured by ISP, Inc.),polyvinylpolypyrrolidone, PVPP and 1-vinyl-2-pyrrolidinone homopolymer.The disintegrants are optionally incorporated in a proportion of about1-15 parts by weight, or about 1-10 parts by weight, or about 3-7 partsby weight, based on 100 parts by weight of the solid formulation.

Solid or liquid buccal or sublingual formulations are useful toadminister a formula 1 compound to a subject (e.g., mammal or human) toachieve a prolonged plasma concentration of the compound. This isaccomplished comprising the steps of (1) preparing a solution of thecompound dissolved in an aqueous carrier solution; (2) disposing thesolution within the subject's sublingual or buccal area in a quantity todeliver a dosage of about 0.01 to about 2.0 or 4.0 mg/kg of body weightor about 0.1-1 mg/kg, e.g., a dose of about 0.1 to about 100 mg or about1-50 mg; and (3) contacting the formulation with the buccal orsublingual mucosa, which creates or maintains prolonged detectableplasma concentrations of the formula 1 compound or a metabolite thereof,e.g., for at least about 2, 4, 8, 24, 48 or 72 hours or more.

In other embodiments, buccal or sublingual delivery of a formula 1compound is accomplished using formulations present as tablets orlozenges, which comprise a candy carrier or a hard candy matrix andsufficient compound, e.g., about 1-100 mg. The candy may be present as asucker or lollipop.

Some embodiments include a solid buccal or sublingual formulationcontaining a formula 1 compound where unit doses of the formulationsubstantially or completely disintegrates or erodes within about 20-120seconds in water at 37° C. or on insertion of the unit dose into thebuccal area or upon placement under the tongue. Such formulations maycomprise a swellable hydrophilic excipient, a water-soluble or awater-dispersible excipient, e.g., one or more of partially hydrolyzedgelatin, hydrolyzed dextran, dextrin, mannitol, alginates, polyvinylalcohol, polyvinyl pyrrolidine, water soluble cellulose derivatives,methylcellulose, ethyl cellulose, carboxymethyl cellulose,hydroxymethylcellulose, hydroxypropyl methylcellulose, microcrystallinecellulose, alginates, gelatin, guar gum, gum tragacanth, gum acacia,polyacrylic acid, polymethacrylic acid, polysilicic acid, polylacticacid, polymaleic acid, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, nonionic blocked polymers, carbomers, polycarbophils, awater soluble starch, dicalcium phosphate, calcium carbonate, silica orpolyethyleneglycol, e.g., PEG2000, PEG8000 or PEG20000, or apolyethylene oxide (“PEO”), PEO100000 or PEO5000000.

Buccal and sublingual formulations comprising a formula 1 compound aresuitable for delivery of the compound to subjects using continuous orintermittent dosing protocols, e.g., any protocol described herein.Excipients disclosed for buccal or sublingual formulations may also beused in formulations suitable for administration by other routesdisclosed herein, e.g., oral or parenteral. Other suitable excipients orformulations that may be modified to comprise a formula 1 compound ormethods to make, use or characterize them have been described, see,e.g., U.S. Pat. Nos. 4,727,064, 4,877,774, 4,764,378, 5,135,752,5,624,677, 5,763,476, 5,958,453, 6,284,262, 6,284,263, 6,264,974,6,248,357, 6,200,593 and 6,103,257.

Other embodiments include the product obtained by storing inventioncompositions or formulations, e.g., unit dosage forms, any ofembodiments (1)-(14) above, or compositions used to make formulations,at about 4-40° C. for at least about 3 days, e.g., storage at ambienttemperature for about 1-24 months. Invention formulations will typicallybe stored in hermetically or induction sealed containers for these timeperiods. Compositions and formulations that comprise a formula 1compound will typically be held in closed or sealed containers,particularly when the composition is a formulation for pharmaceutical orveterinary use. The specification and claims disclose exemplary suitableformulations and unit dosage forms for these embodiments.

Immune modulation. As noted elsewhere, the formula 1 compounds, or thebiologically active substances produced from these compounds byhydrolysis or metabolism in vivo, have a number of clinical andnon-clinical applications. The compounds are generally useful to correctimmune dysregulation, e.g., imbalanced immune responses to diseaseconditions, pathogens or the like, suppression of an innate or acquiredimmune response(s) and inflammation conditions in vertebrate ormammalian subjects, e.g., as disclosed herein. Thus, while the compoundswill generally enhance a deficient immune response in a given clinicalcondition, they will generally reduce the same immune response when itis too active in a different clinical condition. For example, they canenhance insufficient or suboptimal Th1 immune responses, reduce excessor undesirable Th2 immune responses, reduce excess or undesirable Th1immune responses or enhance insufficient or suboptimal Th2 immuneresponses or they can reduce excess or undesirable inflammation or oneor more of its symptoms. The compounds will generally also modulatedysregulated Tc1 and Tc2 immune responses (associated with CD8⁺ T cells)in a similar manner, e.g., excessive Tc1 or Tc2 responses will bedetectably decreased and deficient or suboptimal Tc1 or Tc2 responseswill generally be detectably enhanced.

In modulating one or more activities of Th1, Th2, Tc1 or Tc2 cells ortheir function(s), the formula 1 compounds will typically detectablymodulate one, two, three or more factors, e.g., immune cell subsets orpopulations, cytokines, interleukins, surface antigens such as a CDmolecule(s) and/or their receptors that affect the development,migration, numbers or biological function(s) of such cells. When a Th1or Tc1 cell or population is affected, the formula 1 compounds willtypically increase or decrease the synthesis or level of one, two ormore of an associated effector factor, e.g., IFNγ, IL-2, IL-12, IL-18,T-bet, PPARα and PPARγ or a cell surface molecule, e.g., as disclosedherein or in the cited references, that is associated with or needed fornormal, optimal or enhanced Th1 or Tc1 cells or cell function. Suchmolecules are generally associated with development or enhancement ofTh1 or Tc1 cells or their biological function(s). When a Th2 or Tc2 cellor population is affected, the formula 1 compounds will typicallyincrease or decrease the synthesis or level of one, two or more of anassociated effector factor, e.g., IL-4, IL-5, IL-6, IL-8, IL-10, IL-13,GATA-3, COX-2 or a cell surface molecule, e.g., as disclosed herein orin the cited references, that is associated with or needed for normal,optimal or enhanced Th2 or Tc2 cells or cell function(s). Such moleculesare generally associated with development or enhancement of Th2 or Tc2cells or their biological function(s).

Similarly, when a subject has or is subject to developing an unwanted orexcessive inflammation, the formula 1 compounds will generallydetectably modulate one or more relevant effector factors forinflammation, e.g., a decrease of one, two, three or more of IL-1α,IL-1β, TNFα, MIP-1α, γIFN, IL-6, IL-8, IL-10 and COX-2, or an increaseof one or more suppressor factors or antagonists of inflammation. Suchmodulation can comprise increases or decreases of at least about 2%, 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 100%, 200%, 300%, 500%, 1000%, 5000% or within a range between anytwo of these values, e.g., between about 5-95%, about 10-90%, about5-60% or about 40-95%. In general, such changes leads to a detectableamelioration of an inflammation-associated disease, condition, symptomor to the detectable slowing of the progression thereof or to adetectably reduced incidence or severity of or susceptibility todeveloping an unwanted inflammatory response.

In conditions where an unwanted or excessive Th1, Tc1, Th2 or Tc2response is associated with or causes a disease(s), disease(s)progression, disease(s) state maintenance, condition(s) or symptom(s),the formula 1 compounds will generally decrease the level or one or morebiological activity of one, two or more of their respective associatedeffector molecules. In conditions where a deficient or suboptimal Th1,Tc1, Th2 or Tc2 response is associated with or causes a disease(s),disease(s) progression, disease(s) state maintenance, condition(s) orsymptom(s), the formula 1 compounds will generally increase the level orone or more biological activity of one, two, three or more of theirrespective associated effector molecules. Such changes in the level orbiological activity(ies) the associated effector molecules is generallydetectable using standard methods and is typically an increase (when aresponse is insufficient) or a decrease (when a response is in excess)of at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 98% or within a range between any two of thesevalues, e.g., between about 5-95%, about 10-90%, about 5-60% or about40-95%. In general, such changes leads to a detectable amelioration of adisease, condition, symptom or to the detectable slowing of theprogression thereof or to a detectably reduced incidence or severity ofor susceptibility to developing a disease(s) or the occurrence of asymptom(s) for a at least a portion of subjects that are treated with aformula 1 compound, e.g., at least about 5%, 10%, 20%, 40%, 60% or 80%of treated subjects. The formula 1 compounds may facilitate the clinicalcure of a disease(s), prolong remission of a disease(s) or eliminate orameliorate a clinically detectable symptom(s).

The formula 1 compound will generally also affect the function of otherimmune cell subsets in a similar manner. Thus, when an insufficientmacrophage, dendritic cell or neutrophil response is associated with theestablishment, maintenance or progression of a disease, symptom or acondition, the formula 1 compounds will generally enhance of the levelor a biological activity(ies) of one or more effector moleculeassociated with or needed for an optimal or more normal response orimmune function that is mediated by the macrophages, dendritic cells orneutrophils. Similarly, when the subject suffers from a excessive orpathological activity associated with macrophages, dendritic cells orneutrophils, which is associated with the establishment, maintenance orprogression of a disease, symptom or a condition, the formula 1compounds will generally detectably reduce the level or a biologicalactivity(ies) of one or more effector molecule associated with or neededfor an optimal or more normal response or immune function that ismediated by the macrophages, dendritic cells or neutrophils. Sucheffector molecules are as described herein or in the cited references.

As used herein, reference to Th1 or Th2 immune responses means suchresponses as observed in mammals generally and not as observed in themurine system, from which the Th1 and Th2 terminology originated. Thus,in humans, Th1 cells are CD4+T lymphocytes and they usuallypreferentially display chemokine receptors CXCR3 and CCR5, while Th2cells are CD4+T lymphocytes and usually preferentially express the CCR4,CCR8 and/or CXCR4 chemokine receptor molecule(s) and generally a smalleramount of CCR3, see, e.g., U. Syrbe et al., Springer Semin.Immunopathol. 1999 21:263-285, S. Sebastiani et al., J. Immunol. 2001166:996-1002. Tc1 and Tc2 immune responses are mediated by CD8⁺lymphocytes and means to identify these cells and their associatedlymphokines, cell specific antigens and biological activities have beendescribed, see, e.g., M. B. Faries et al., Blood 2001 98:2489-2497, W.L. Chan et al., J. Immunol. 2001 167:1238-1244, C. Prezzi et al., Eur.J. Immunol. 2001 31:894-906, H. Ochi et al., J. Neuroimmunol. 2001119:297-305, D. H. Fowler and R. E. Gress, Leukemia and Lymphoma 200038:221-234.

The formula 1 compounds are useful in reestablishing normal immunefunction in various immune dysregulation or immune suppressionconditions. For example, they are useful to treat, slow progression ofor to ameliorate one or more symptoms associated with one or more of anautoimmune condition(s), a inflammation condition(s), an infection(s), acancer(s), a precancer(s), a chemotherapy(ies), radiation therapy, aburn(s), a trauma(s), a surgery(ies), a pulmonary condition, acardiovascular disease(s) and a neurological or neurodegenerativedisease(s). Without being limited to any theory, the formula 1 compoundsare believed to act through several mechanisms, including by directly orindirectly modulating steroid receptor activity or by affecting ormodulating other biological targets such as transcription factors,steroid binding proteins or enzymes in at least some of the diseases,conditions or symptoms disclosed herein.

The formula 1 compounds are useful to modulate delayed-typehypersensitivity (“DTH”) responses and anergic conditions in subjectshaving to subject to developing abnormal DHT responses or anergy. Meansto measure such responses and conditions are known and can be used tocharacterize the effects of the formula 1 compounds on these responsesand conditions. See, e.g., A. E. Brown, et al., J. Med. Assoc. Thailand83:633-639 2000, R. A. Smith et al., J. Adolesc. Health 27:384-390 2000,N. M. Ampel, Med. Mycology 37:245-250 1999. The compounds will generallydetectably enhance or restore DTH in immune suppression conditions. Theywill also generally detectably reduce or eliminate anergy in subjectshaving significantly reduced or no immune response to, e.g., specificantigens or pathogens.

The invention provides a method to detectably enhance an antigenspecific immune response, cell mediated immune response or adelayed-type hypersensitivity immune response in a subject havingimpaired or negligible antigen specific immune response, cell mediatedimmune response or delayed-type hypersensitivity immune response,comprising administering to the subject, or delivering to the subject'stissues, an effective amount of a formula 1 compound. The antigenspecific immune response, cell-mediated immune response or delayed-typehypersensitivity immune response can be enhanced at least about 25%, atleast about 40%, at least about 50%, at least about 60%, at least about75% or at least about 90%. Some of the subjects may have an antigenspecific immune response, cell mediated immune response or adelayed-type hypersensitivity immune response that is impaired ornegligible, e.g., about 50% or less or about 30% or less or about 10% orless of the response that an otherwise normal subject would be expectedto have. Such subjects may not detectably respond to at least 1 antigenout of 2, 3, 4 or 5 antigens that a normal subject would respond to. Insome embodiments, the subject is an HIV-infected human having a CD4+Tcell count of about 0-150 cells/mm³ or about 2-100 cells/mm³ and/orwherein the antigen specific immune response, cell mediated immuneresponse or delayed-type hypersensitivity immune response is an enhancedresponse to a viral, bacterial, parasite or fungal antigen such as anHIV, HCV, HBV or CMV antigen such as a viral or HIV core antigen or HIVp24 antigen or a viral or HIV envelope antigen, a Candida antigen, aviral, bacterial, parasite or fungal antigen essentially as describedherein or to phytohemagglutinin. The responses to treatment with aformula 1 compound may be quantitated by, e.g., mixed lymphocytereaction, ELIspot analysis or flow cytometric analysis of, e.g.,circulating blood cells such as CD4⁺ or CD8⁺ T cells or for levels ofcytokines (e.g., IL-2, TNFα or IFNγ) in such cells. Such analyses havebeen described, e.g., V. P. Badovinac and J. T. Hardy, J. Immunol.Methods 2000, 238:107-117, N. Favre et al., J. Immunol. Methods 1997,204:57-66, E. Hagiwara et al., Cytokine 1995, 7:815-822, N. W. Lukacs etal., Blood 1993, 82:3668-3674, M. Umemoto et al., Clin. Exp. Immunol.1998, 112:459-463, A. Fietta et al., Gerontology 1994, 40:237-245, C. H.Orteu et al., J. Immunol. 1998, 161:1619-1629.

Clinical indications that have an association with or have a symptom(s)that is consistent or associated with an excessive or unwanted Th2immune response include, e.g., fatigue, pain, fever or an increasedincidence of infection, schizophrenia, acute myelitis, tumorprogression, progressive systemic sclerosis, Omenn's syndrome, atopicdisease, atopy, allergen hypersensitivity, atopic asthma, atopicdermatitis, burns, trauma (e.g., bone fracture, hemorrhage, surgery),immune responses to xenotransplantation, chronic periodontitis, SLE(systemic lupus erythematosus), discoid lupus erythematosus,osteoporosis, myasthenia gravis, Graves disease, mite-associatedulcerative dermatitis, rheumatoid arthritis and osteoarthritis.Excessive Th2 immune responses are also associated with an unwantedsymptom or pathology, e.g., fatigue, pain, fever or an increasedincidence of infection, that is associated with aging, allergy andinflammation conditions such as allergic bronchopulmonary aspergillosisin cystic fibrosis patients, allergic respiratory disease, allergicrhinitis, atopic dermatitis, subepithelial fibrosis in airwayhyperresponsiveness, chronic sinusitis, perennial allergic rhinitis,fibrosing alveolitis (lung fibrosis). This common underlying immunecomponent is at least part of the pathology or symptoms of all of theseconditions. This allows a formula 1 compound to be effectively used toprevent or treat the condition or to treat or ameliorate one or moresymptoms that are associated with these conditions. Thus, in someembodiments, an unwanted or excessive Th2 response is present andamelioration of one or more symptoms associated with this condition isaccomplished by administering an effective amount of a formula 1compound according to the methods described herein, e.g., formula 1compound is administered using a formulation and a route ofadministration essentially as described herein on an intermittent or adaily basis.

Typically, unwanted Th2 immune responses are associated with, or causedby, increased expression of one or more cytokines or interleukins suchas one, two, three or more of cortisol, IL-4, IL-5, IL-6, IL-10 andIL-13. Administration of a formula 1 compound will generally reduce theexpression of one or more of the Th2-associated cytokines orinterleukins. At the same time, the compounds generally enhance theexpression of one or more cytokines or interleukins associated with Th1immune responses. Because of their capacity to modulate or to balanceTh1 and Th2 immune responses, the compounds are useful for a variety ofclinical conditions, e.g., infection, immunosuppression or cancer, wherean enhanced Th1 immune response is desired. Effects of the formula 1compounds in treating, preventing or slowing the progression of theclinical conditions described herein can include one or more of (1)enhancing the Th1 character of a subject's immune response or immunestatus, (2) increasing the intensity of a Th1 or a Th2 immune responseor both and (3) decreasing inflammation or a symptom thereof.

Exemplary conditions where an immune imbalance or an excessive Th1immune response is involved include autoimmune diseases such as multiplesclerosis, Crohn's disease (regional enteritis), ulcerative colitis,inflammatory bowel disease, rheumatoid arthritis, reactive arthritis,acute allograft rejection, sarcoidosis, type 1 diabetes mellitus,Helicobacter pylori associated peptic ulcer, graft versus host diseaseand Hashimotos' thyroiditis. Because these conditions are associatedwith a similar type immune dysfunction, a formula 1 compound can beeffectively used to prevent or treat these conditions or to treat orameliorate one or more symptoms associated therewith. Thus, in someembodiments, an unwanted or excessive Th1 response is present andamelioration of one or more symptoms associated with this condition isaccomplished by administering an effective amount of a formula 1compound according to the methods described herein, e.g., formula 1compound is administered using a formulation and a route ofadministration essentially as described herein on an intermittent or adaily basis. In other embodiments, an deficient Th1 response isenhanced, which is optionally observed as a detectable increase in oneor more of IFNγ, IL-2, IL-12 or IL-18 in Th1 cells or in accessory cellssuch as a dendritic cell or macrophage. In all of the conditions wherean insufficient or excess Th1, Th2, Tc1 or Tc2 response is present,amelioration of one or more symptoms associated with the condition isaccomplished by administering an effective amount of a formula 1compound according to the methods described herein.

Aspects of the invention include the use or administration ofcompositions or formulations that comprise a carrier and an amount of atleast one formula 1 compound effective to detectably modulate an immuneparameter. For example, to enhance the relative proportion of a desiredimmune cell subset, e.g., CD4⁺ T cells, CD8⁺ T cells, NK cells, LAKcells, neutrophils, granulocytes, basophils, eosinophils or dendriticcells, or to modulate (detectably increase or decrease) one or morefunctions of immune cell subsets. The formula 1 compounds can modulatethe expression of CD molecules or alter the proportion of cell subsets,e.g., CD4⁺ or CD8⁺ T cells, or their relative numbers in a subject'sblood or tissues. CD and related molecules participate in the functionof various immune cell subsets and can be useful as markers for immunefunction in vivo. In some aspects, the formula 1 compounds activateimmune cells which generally alters (increases or decreases) expressionof, or changes the numbers of cells that express one or more of, CD4,CD6, CD8, CD25, CD27, CD28, CD30, CD36, CD38, CD39, CD43, CD45RA,CD45RO, CD62L, CD69, CD71, CD90 or HLA-DR molecules. Often, the numbersof cells that express these molecules are increased, e.g., CD25, CD16 orCD69. Typically, such increases are observed as an increased proportionof circulating white blood cells that express one or more of thesemolecules or white blood cells CXCR3, CCR5, CCR4, CCR8 and/or CXCR4. Insome cases the number of such molecules per cell is detectably altered.

Expression of one or more adhesion molecules CD2, CD5, CD8, CD11a,CD11b, CD11c, CD18, CD29, CD31, CD36, CD44, CD49a, CD49b, CD49c, CD49d,CD49e, CD49f, CD50, CD54, CD58, CD103 or CD104 are also detectablymodulated after administration of the formula 1 compounds to a subject.Often, the numbers of cells that express these molecules are increased,e.g., CD5 or CD56. The adhesion molecules function in various aspects ofimmune responses, such as binding to class I MHC molecules, transducingsignals between cells or binding to molecules in the extracellularmatrix associated with endothelial or other cell types. Administrationof the formula 1 compounds to a subject also affects the numbers ofcertain immune cell subsets, e.g., NK cells (e.g., CD8⁻, CD56⁺ or CD8⁺,CD56⁺) or lymphokine activated killer cells (LAK). Increased circulatingNK or LAK cells are typically observed, which is reflected in increasednumbers of cells that express one or more of CD16, CD38, CD56, CD57 orCD94. Also, increased numbers of circulating dendritic cell precursorsare observed, as shown by increases in cells that express one or more ofCD11c, CD80, CD83, CD106 or CD123. Although one can observe an increasedproportion of circulating white blood cells that express one or more ofthese molecules, in some instances the number of such molecules per cellis detectably altered. Both the cell numbers and the density of CDmolecule per cell can also be detectably modulated. Modulation of immunecell subsets typically occurs on intermittent dosing of a formula 1compound, but will arise from any suitable dosing regimen, e.g., asdescribed herein.

Expression of one or more homing or other receptors or receptor subunitssuch as CD62L, CLA-1, LFA1, CD44, ICAM, VCAM or ECAM may also bedetectably affected after administration of the formula 1 compounds to asubject. The numbers of cells that express these molecules, or therelative amounts per cell of, e.g., CD44 or CD62L, may be increasedwhere a desired immune response is desired, e.g., migration of T cellsto mucosal tissues or exposure of naïve T cells to antigen in lymphnodes. Alternatively, numbers of cells that express these molecules, orthe relative amounts per cell of, e.g., CLA-1, may be decreased whereinhibition of an undesired immune response, such as an inflammatoryresponse is desired. The subject's response to such enhanced expressionincludes migration of cells such as movement of naïve T cells toperipheral lymph nodes in response to modulation of CD62L or otherhoming receptor expression. Thus, the formula 1 compounds can alsofacilitate migration of various immune cell types, e.g., dendriticcells, NK cells, LAK cells, macrophages or lymphocytes, from onelocation to another within a subject. For example, the compounds canenhance dendritic cell or lymphocyte migration from areas such as theskin tissues to the gut associated lymphoid tissue (“GALT”), lymph nodesor spleen. Such migration may facilitate the function of those celltypes by increasing their transit to tissues where their effectorfunctions, e.g., antigen presentation by dendritic cells, normallyoccur. The migration period is often relatively transient (e.g.,observable over about 1-7 days) or occasionally longer (e.g., occurringfor about 8-40 days), depending on the dosing regimen and other factors.This migration can be observed by standard methods, e.g., by cellstaining, by PCR analyses or by determining the presence of a given celltype in circulation or determining a decrease in the number circulatingcells. A decrease would generally reflect sequestration of an immunecell population(s) in a tissue(s) where the immune cell normallyexercises its effector functions.

Thus, in some embodiments, the migration of one or more immune cellsubsets such as CD11C⁺ cells from tissue such as skin or lung throughthe blood to immune tissue such as lymph nodes or GALT is seen as atransient increase in the level of circulating CD11C⁺ cells in responseto exposure of the subject's tissues to a suitable amount of a formula 1compound. Thus, the level of CD11C⁺ cells in the blood will generallydetectably increase, e.g., a statistically significant increase, plateauand then decrease as migration of the cells to immune tissue subsides.In these embodiments, the proportion of the cells of the affected immunecell subset is typically relatively low in most physiological immunestates, e.g., normal or abnormal immune conditions, compared to thetotal white blood cell population in circulation. In other embodiments,the migration of one or more immune cell subsets such as CD123⁺ cellsfrom the circulation to immune tissue such as lymph nodes or GALTresults in a decrease. In these embodiments, the decrease in the numbersof circulating immune cells reflects the migration of the immune cellsfrom the blood to immune tissue such as lymph nodes or GALT. Such adecrease may be transient and followed by recovery of the affectedimmune cell subset(s) over about 2 to 24 weeks. In conducting theseembodiments, administration of the formula 1 compound to the subject isaccomplished using the formulations or the methods as described herein.

Thus, an aspect of the invention is a method to enhance the migration ofone or more immune cell types in a subject from one location (e.g., bonemarrow, circulating blood or a tissue such as the skin, liver, centralnervous system or lung) to another (e.g., to the blood or to a lymphoidtissue such as a lymph node, spleen or a mucosal tissue such as GALT) byadministration to a subject as described herein of an effective amountof a formula 1 compound essentially as described by any of the methodsdisclosed herein. A related aspect is the monitoring, e.g., by suitableblood counts or tissue biopsy, of the subject's response to determinethe timing and extent of such immune cell migration.

Other CD molecules that are modulated by the presence of the formula 1compounds in a subject include cytokine receptor molecules such as oneor more of CD115, CDW116, CD117, CD118, CDW119, CD120a, CD120b, CD121a,CD121b, CD122, CD123, CD124, CD125 CD126, CDW127, CDW128 or CDW130.Often, the numbers of receptor molecules per cell will be modulated. Forexample, receptors for cytokines that mediate or facilitate Th1 immuneresponses or innate immune responses (e.g., one or more of IL-1α, IL-1β,IL-2, IL-4, IL-12, γIFN or α-interferon) will typically increase in oron cells that mediate Th1 or innate immune responses. Modulation ofthese molecules may be by direct interactions with a receptor(s) in thecell that expresses the cytokine receptor or indirectly by modulation ofcytokine synthesis in the affected cells or in other cells, typicallyimmune cells that may interact with the cells whose receptor synthesisis being modulated. Thus, autocrine or paracrine mechanisms may underliesome of the effects associated with administration of a formula 1compound(s) such as altered cytokine profiles in immune cells or alteredimmune cell populations. Endocrine cytokine mechanisms may alsocontribute to desired immune responses.

Treatment of a subject with a formula 1 compound can result in a changeof at least about 20-80% or about 25-50% above or below (e.g., at least30% or at least 40% above or below) the control or basal level ofaffected immune cell subsets. For example, increases of more than about30% in the total numbers of activated CD8⁺ T cells, e.g., CD8⁺, CD69⁺,CD25⁺ T cells, CD8⁺, CD69⁺, CD25⁻ T cells or CD8+, CD69⁻, CD25⁺ T cells,can occur by 7 days after a single dose of a formula 1 compound to asubject. Such increases may be greater than 50%, 60% or 100% in thetotal numbers of activated CD8⁺ T cells or subsets of activated CD8⁺ Tcells in individual subjects. Typically such increases are about in thetotal numbers of activated CD8⁺ T cells or subsets of activated CD8⁺ Tcells averages about 30-40%, with individual subjects experiencingincreases over 100% in the numbers of activated CD8⁺ T cells per unitblood volume compared to the basal level.

Administration of the formula 1 compounds can affect other immune cellsubsets. For example, the concentration of circulating CD4⁺, CD69⁺,CD25⁻ (Th1 helper cells) and CD8⁺, CD16⁺, CD38⁺ LAK cells or CD8⁻,CD16⁺, CD38⁺ LAK cells typically increases during or after the course ofdosing a subject with a formula 1 compound. Also, CD8⁻, CD16⁺, CD38⁺ andCD8⁺, CD16⁺, CD38⁺ (ADCC effector cells) and low side scatter Lin⁻, DR⁺,CD123⁺ (dendritic precursors) or low side scatter Lin⁻, DR⁺, CD11c⁺(dendritic cells or precursors) may show modest to significantincreases.

In subjects that are immunosuppressed, e.g., from certain infections(e.g., viral (HIV, HCV), bacterial infection or parasite infection) orfrom chemotherapy (e.g., an antiviral therapy, a cancer chemotherapy ora radiation therapy), administration of the formula 1 compounds to thesubject results in a favorable shift in the balance of Th1 or Th2responses the subject can mount in the face of immunosuppression. WhenTh1 responses are suboptimal or insufficient, treatment with a formula 1compound results in enhancement of Th1 responses or a reduction in Th2responses. Conversely, when Th2 responses are suboptimal orinsufficient, treatment with a formula 1 compound results in enhancementof Th2 responses, which may occur with a concomitant modulation(increase or decrease) in Th1 responses. The formula 1 compounds canthus be used to shift the nature of a subject's immune response toresult in a more balanced immune response from immunosuppression.Alternatively, the compounds can selectively suppress inappropriate orunwanted immune responses. Enhanced Th1 responses appears to be at leastpartly due to one or more of (i) a reduction in biological restraints,e.g., high levels of IL-4 or IL-10, on Th1 functions by preexistingprimed Th1 effector cells, (ii) enhanced differentiation of Th0 cells toTh1 cells or enhanced responses mediated by Th1 cells, (iii) enhancedfunction of accessory cell function, e.g., antigen presentation bydendritic cells, dendritic precursor or progenitor cells or bymacrophages or their precursors or progenitors, (iv) enhancedproliferation and differentiation of Th1 precursor or progenitor cells,(v) enhanced IL-12 expression in dendritic cells or their precursors,which results in enhanced differentiation of Th1 cells from Th0precursors, (vi) enhanced expression or activity of factors associatedwith Th1 functions, e.g., IL-2, gamma interferon (γIFN or IFNγ), IL-18or lymphotoxin.

An aspect of the invention methods is an alteration in the expression ofIL-4 or IL-10 that occurs after administration of a formula 1 compound,e.g., BrEA, to a subject. A consistent observation is that extracellularIL-4 or IL-10 levels rapidly decrease to levels that are undetectable byELISA. Intracellular IL-10 levels are reduced to levels that are near orbelow the limits of detection by flow cytometry. The administration of aformula 1 compound to a subject thus provides a means to inhibit eitheror both of these interleukins. Such inhibition may be associated withenhancement of Th1 immune responses relative to Th2 or Th0 responses,e.g., in subjects where Th1 responses are suppressed (e.g., from viral,bacterial or parasite infection (HIV, HCV, etc) or chemotherapy) or areotherwise suboptimal. In many subjects, levels of either IL-4 or IL-10,usually IL-10, before dosing with a formula 1 compound is low orundetectable. In these subjects, dosing with the formula 1 compoundresults in a rapid drop in the interleukin that is detectable, usuallyIL-4.

Clinical conditions are described in more detail below where the formula1 compounds are useful for treating, preventing, slowing the progressionof, or ameliorating one or more symptoms associated with the describedconditions. In any these conditions, any formula 1 compound disclosedherein can be used according to one or more of the dosing methods thatare disclosed herein. For these conditions, dosages for the formula 1compounds, formulations and routes of administration are as describedherein. Additional information regarding these and other clinicalconditions or symptoms that can be treated, prevented or amelioratedwith the formula 1 compounds are found at e.g., The Merck Manual,17^(th) edition, M. H. Beers and R. Berkow editors, 1999, Merck ResearchLaboratories, Whitehouse Station, N.J., ISBN 0911910-10-7, or in otherreferences cited herein.

Responses to treatment of a subject having a condition disclosed hereinwith a formula 1 compound is optionally monitored by observing changesin one or more immune or other appropriate clinical parameters, e.g., asdescribed herein or in D. S. Jacobs et al., editors, Laboratory TestHandbook, 4^(th) edition, pages 11-686, Lexi-Comp Inc., Hudson, Ohio,ISBN 0-916589-36-6, or in any of the references cited herein, or bymonitoring the progression or severity of the underlying conditionaccording to known methods, e.g., J. B. Peter, editor, Use andInterpretation of Laboratory Tests in Infectious Disease, 5^(th)Edition, pages 1-309, 1998, Specialty Laboratories, Santa Monica,Calif., ISBN 1-889342-13-0.

Infection treatments. In some embodiments, the formula 1 compound(s) isadministered to a subject who has a pathogen infection, such as a viral,bacterial, fungal, yeast, intracellular parasite or extracellularparasite infection. The formula 1 compounds can be considered for use ina broad scope of infections (see, e.g., J. B. Peter, editor, Use andInterpretation of Laboratory Tests in Infectious Disease, 5^(th)edition, Specialty Laboratories, Santa Monica, Calif. 90404, 1998, pages1-271), since the compounds generally enhance Th1 immune responsesand/or reduce Th2 immune responses and/or reduce inflammation or itssymptoms. Difficulty in treating many infections, e.g., progressivetoxoplasmic encephalitis, malaria, tuberculosis, Leishmaniasis andschistosomiasis, often appear to be associated with one or more of anunwanted Th2 immune responses, a suboptimal Th1 response or thedevelopment of resistance of the infectious agent to antimicrobialagents. For example, in disseminated or diffuse tuberculosis, a reducedTh2 response would be desirable to allow a patient to slow progressionof the disease or to clear infected cells more efficiently. In treatingchloroquine resistant or sensitive malaria, the formula 1 compounds haveessentially the same activity.

Exemplary viral infections that the formula 1 compounds can be used totreat, prevent or ameliorate include infections by one or more DNA orRNA viruses, or a symptom(s) associated with such infection(s), such asa genogroup, Glade, serotype, serotype subtypes, isolate, strain,subtype or so forth of influenza viruses (e.g., a human influenza Avirus, a human influenza B virus, an avian (e.g., chicken, duck, goose)influenza virus, a swine influenza virus or a recombinant avian-swineinfluenza virus), respiratory syncytial viruses, Rotaviruses,Hantaviruses, animal or human Papillomaviruses (e.g., HPV-16, HPV-18),Poxviruses, Poliovirus, rabies viruses, human and animal Retroviruses(e.g., HIV-1, HIV-2, LAV, human T-cell leukemia virus I (“HTLV I”), HTLVII, HTLV III, SIV, SHIV, FIV, FeLV), Togaviruses and Flaviviruses (e.g.,West Nile Virus, Yellow Fever Virus, Dengue viruses), Herpesviruses(e.g., CMV, EBV, Varicella Zoster Virus, Herpes simplex virus 1(“HSV-1”), Herpes simplex virus 2 (“HSV-2”), human Herpesvirus 6(“HHV-6”), human Herpesvirus 8 (“HHV-8”)), measles viruses, mumpsviruses, rubella virus, Hepadnaviruses or hepatitis viruses,Adenoviruses, Retroviruses, Togaviruses, Alphaviruses, Arboviruses,Coronaviruses, Flaviviruses, Filoviruses, Rhinoviruses, Picornaviruses,Papovaviruses, Bunyaviruses, Picornaviruses, Poxviruses and/orPestiviruses.

Specific viruses, including their genogroups, clades, isolates,serotypes, serotype subtypes, strains and so forth, that may establish avirus infection susceptible to the treatment methods disclosed hereininclude one or more of human hepatitis C virus (“HCV”), human hepatitisB virus (“HBV”), human hepatitis A virus (“HAV”), human hepatitis deltavirus, human hepatitis E virus, duck hepatitis virus, woodchuckhepatitis virus, one or more of human herpesviruses 1, 2, 3, 4, 5, 6A,6B, 7 or 8, one or more of human papilloma viruses 1-60, e.g., HPV 6,HPV 11, HPV 16, HPV 18, HPV 31, HPV 45, animal papilloma viruses,poliovirus 1, poliovirus 2, poliovirus 3, one or more of Dengue virustypes 1, 2, 3 or 4, one or more of foot-and-mouth disease virus 1-7,including serotypes 0, A, C, SAT 1, SAT 2, SAT 3 and ASIA 1, one or moreof coxsackievirus A1-A22, A24, and B1-B6, one or more of human echovirus1-9, 11-27 and 29-34, one or more of human enterovirus 68-71, one ormore of adenovirus 1-49, one or more of Parainfluenza viruses 1, 2, 3 or4, Human respiratory coronaviruses 229E and OC43, one or more of Humanrotaviruses, BK virus, Bunyamwera virus, California Encephalitis Virus,Central European Encephalitis Virus, encephalomyocarditis virus,Colorado tick fever virus, Cowpox virus, Eastern equine encephalitisvirus, Venezuelan equine encephalitis virus, Argentine hemorrhagic fevervirus, Bolivian hemorrhagic fever virus, Lacrosse virus, Hantaan virus,JC virus, Lassa virus, Lymphocytic choriomeningitis virus, Kyasanurforest virus, Marburg virus, Measles virus, Mokola virus, Monkeypoxvirus, Molluscum contagiosum virus, Mumps virus, Murray Valleyencephalitis virus, Norwalk virus, O'nyong-nyong virus, Omsk hemmorhagicvirus, Orf virus, Rabies virus, RA-1 virus, Western equine encephalitisvirus, Japanese encephalitis virus, Yellow Fever Virus, West Nile virus,Variola (smallpox) virus, cowpox virus, Vaccinia virus, Ebola virus,Respiratory syncytial virus, human cytomegalovirus, Rhinoviruses 1-113,Rift Valley fever virus, Ros river virus, Rubella virus, Russianspring-summer encephalitis virus, Sandfly fever viruses, St. Louisencephalitis virus, SV40 virus, vaccinia virus, Varicella-zoster virus,Vesicular stomatitis viruses and Bovine Viral Diarrhea Virus. Exemplaryviruses have been described. See, for example B. N. Fields, et al.,editors, Fundamental Virology, 3^(rd) edition, 1996, Lippencott-RavenPublishers, see chapter 2 at pages 23-57, including table 4 at pages26-27, table 5 at pages 28-29, chapter 17 at pages 523-539, chapters26-27 at pages 763-916, chapter 32 at pages 1043-1108 and chapter 35 atpages 1199-1233.

In an exemplary embodiment, human patients infected with HCV are dosedwith an aqueous isotonic α-cyclodextrin or β-cyclodextrin, e.g.,hydroxypropyl-6-cyclodextrin, formulation containing about 20 mg/mLBrEA. The formulation is delivered intravenously in a single daily doseor two subdoses per day. The patients are dosed with 1 to 10 mg/kg/dayfor 4 to 10 days, followed by no dosing for 5 to 30 days, followed bydosing again with the cyclodextrin formulation for 4 to 10 days. Thedosing regimen is repeated one, two or more times. Clinical markers forHCV infection are followed during treatment, e.g., viral nucleic acid inthe blood or plasma, liver enzyme levels in the blood or plasma (e.g.,AST/SGOT, ALT/SGPT, alkaline phosphatase). For these patients, ananti-HCV treatment(s), e.g., γIFN, αIFN, a retroviral proteaseinhibitor, a nucleoside analog, and/or ribavirin, is optionally startedor continued according to the recommendations of the patient's doctorand with the patient's informed approval. In some of these embodiments,a formula 1 compound(s) is administered daily continuously as acomponent in an oral or parenteral composition or formulation, e.g., fora formula 1 compound(s) that is a new compound per se. BrEA isoptionally also administered systemically using, e.g., a parenteralformulation to deliver 0.1-5 mg/kg/day either daily or every other dayfor about 1 to 4 months, or an oral formulation to deliver about 0.5-40mg/kg/day either daily or every other day for about 1 to 4 months.

In related embodiments, the formula 1 compounds are used to treat,prevent or ameliorate Arbovirus infections, Arenavirus infections,Hantavirus infections and hemorrhagic fever virus infections, or asymptom(s) or complication(s) thereof, in subjects such as humans. Intreating such infections, the formula 1 compound is administered at adosage disclosed herein, e.g., about 0.5-4 mg/kg/day by buccal deliveryor by a parenteral route such as subcutaneous or intramuscularinjection, for about 5-14 consecutive days. An oral dosage would beabout 10-25 mg/kg/day of a formula 1 compound for about 5-14 consecutivedays. Typically dosing with the formula 1 compound will begin at thetime that (or shortly thereafter, e.g., within about 1-12 hours) theinfection is suspected or is diagnosed. The patient is optionallymonitored and the amelioration of one or more symptoms or a sloweddisease progression is observed. For example, arbovirus encephalitis,which is typically a serious brain infection, can be treated, preventedor ameliorated. Several types of viral encephalitis are transmitted byinsect bites, e.g., western equine encephalitis, eastern equineencephalitis, St. Louis encephalitis, and California encephalitis. Inthese infections the formula 1 compounds can treat, prevent orameliorate one or more symptoms including fever, headache, drowsiness,vomiting, stiff neck, mental confusion, muscle trembling, convulsions,and coma. Hemorrhagic fevers in humans are associated with infection byHantaviruses and Filoviruses such as Ebola and Marburg viruses, whichcan cause infections that include Korean, Bolivian and Argentineanhemorrhagic fevers, Congo fever and Lassa fever. The formula 1 compoundscan be used to treat, prevent or ameliorate one or more symptoms ofthese infections, e.g., a hemorrhagic fever virus infection, in asubject such as a human. Ebola virus and Marburg virus infections can beaccompanied by mucous membrane bleeding, fever, diarrhea, bleeding,myalgia, lymphadenopathy, pain, e.g., chest pain, coughing, vomiting,stupor, coma or loss of consciousness and failure of multiple organs. Inthese embodiments, the formula 1 compounds can function by one or moremechanisms, including enhancing innate immune responses, modulating,e.g., detectably increase or decrease, the level or activity of one ormore of IL-1α, IL-1β, TNFα, IL-6, IL-8, IL-10, gro-α, IFN-γ, MCP-1,MIP-1α, MIP-1β, IP-10, GM-CSF, RANTES or their isotypes or homologs orcortisol. The compounds can also detectably increase the synthesis orlevel of IFN-α, IFN-β, IgG1 and/or IgG3 in these conditions.

Treatment of a subject such as a human who is anticipated to potentiallycome in contact with Arbovirus, Arenavirus, Hantavirus or a hemorrhagicfever virus is accomplished by administering a formula 1 compound to thesubject by, e.g., daily or intermittent dosing, beginning at about 1-14days before an anticipated potential exposure. The daily doses androutes of administration are essentially as described herein, e.g., foran adult human, about 0.5-4 mg/kg/day by buccal delivery or by aparenteral route such as subcutaneous or intramuscular injection for 1,2, 3, 4, 5, 6, 7 or 8 consecutive days or on alternate days over a 4, 6,8, 10, 12 or 14 day period in advance of a potential exposure.

Hantavirus infection is a viral disease that rodents can transmit tohumans and the infection is associated with serious lung or kidneyinfection. Symptoms of Hantavirus infection of the lungs include one ormore of fever, muscle pain, myalgia, headache, abdominal pain,conjunctival bleeding, diarrhea, vomiting, coughing, shortness of breathor low blood pressure (shock). Hantavirus kidney infection may be mildor severe and is associated with fever, headache, backache, abdominalpain, small bruise-like patches on the whites of the eyes, abdominalrash, impaired kidney function, nausea, loss of appetite, fatigue andintracranial bleeding.

The formula 1 compounds can also be used to treat, prevent or ameliorateinfections caused by members of the Poxyiridae family, e.g., members ofthe Orthopoxvirus genus in subjects such as mammals or humans. Thecompounds can be used to treat, ameliorate or prevent one or moresymptoms associated with Orthopoxvirus infections. For example, thevariola or smallpox virus causes a serious infection with symptoms thatinclude fever, chills, backache, headache, skin lesions and death. Intreating Orthopoxvirus infections, the formula 1 compound isadministered at a dosage disclosed herein, e.g., about 0.5-4 mg/kg/dayby buccal delivery or by a parenteral route such as subcutaneous orintramuscular injection, for about 5-14 consecutive days. Typicallydosing with the formula 1 compound will begin at the time that (orshortly thereafter, e.g., within about 1-12 hours) the infection issuspected or is diagnosed. In treating Orthopoxvirus infections such asa variola infection, the formula 1 compounds can result in enhancedefficacy of host factors such as CC cytokines or interferons such asIFN-α or IFN-γ. The subject may also be optionally treated with anotheragent such as IFN-γ, a nucleoside analog or a nucleotide analog such asone described herein or in the cited references.

Treatment of a subject such as a human who is anticipated to potentiallycome in contact with an Orthopoxvirus such as the variola virus or thevaccinia virus is accomplished by administering a formula 1 compound tothe subject by, e.g., daily or intermittent dosing, beginning at about1-14 days before an anticipated potential exposure. The daily doses androutes of administration are essentially as described herein, e.g., foran adult human, about 0.1-10 mg/kg/day by buccal delivery or by aparenteral route such as subcutaneous or intramuscular injection for 1,2, 3, 4, 5, 6, 7 or 8 consecutive days or on alternate days over a 4, 6,8, 10, 12 or 14 day period in advance of a potential exposure.

In other embodiments, formula 1 compound(s) are administered to asubject or delivered to the tissues of a subject who has a pathogeninfection (or is susceptible to an infection) such as one caused by orassociated with a parasite, bacterium, fungus or yeast, to slow theprogression of infection, interfere with replication or development ofthe infectious agent or to ameliorate one or more of the associatedsymptoms, e.g., weight loss, anemia, fever, pain, fatigue, inflammation,immune dysfunction, secondary infections, skin lesions or ulcers or moodchanges such as depression. Parasites include malaria parasites,sleeping sickness parasites and parasites associated withgastrointestinal infections.

Exemplary parasite, fungi, yeast and bacterial infections that can betreated, prevented or ameliorated in subjects such as mammals or humans,include ones caused by or associated with species, groups, genotypes,serotypes, strains or isolates of gastrointestinal helminths,microsporidia, isospora, cryptococci, cryptosporidia (Cryptosporidiumparvum), Trypanosoma sp. (e.g., T. brucei, T. gambiense, T. cruzi, T.evansi), Leishmania sp. (e.g., L. donovani, L. major, L. braziliensis),Plasmodium sp. (e.g., P. falciparum, P. knowlesi, P. vivax, P. berghei,P. yoelli), Ehrlichia sp. (e.g., E. canis, E. chaffeensis, E.phagocytophila, E. equi, E. sennetsu), Entamoeba sp., Babesia microti,Bacillus anthracis, Brucella sp. (e.g., B. militensis, B. abortus),Bartonella sp. (B. henselae), Bordetella sp. (e.g., B. bronchiseptica,B. pertussis), Enterococcus sp. (e.g., E. faecalis, E. faecium),Enterobacter sp., Erysipelothrix rhusiopathiae, Escherichia sp. (E.coli), Haemophilus sp. (e.g., H. somnus, H. influenzae, H.parainfluenzae), Klebsiella sp. Legionella pneumonia, Listeria (e.g., L.monocytogenes, L. ivanovii), Morganella sp. (e.g., M. morganii),Mycobacterium sp. (e.g., M. avium, M. bovis, M. leprae, M. tuberculosis,M. pneumoniae. M. penetrans), Mycoplasma sp. (e.g., M. fermentans, M.penetrans, M. pneumoniae), Neisseria (e.g., N. gonorrhoeae, N.meningitidis), Nocardia asteroides, Proteus sp. (e.g., P. mirabilis, P.vulgaris, P. myxofaciens), Providencia sp. (e.g., P. rettgeri, P.stuartii), Pseudomonas sp. (P. aeruginosa), Salmonella sp. (e.g., S.typhimurium, S. tyhpi, S. paratyhpi, S. dublin, S. enteritidis, S.schottmuelleri, S. hirschfeldii), Serratia sp., Shigella sp. (e.g., S.flexneri, S. sonnei, S. dysenteriae), Streptococcus sp. (e.g., S.pneumoniae, S. pyogenes, S. faecalis, S. faecium, S. agalactiae, S.mutans, S. sanguis), Staphylococcus sp. (e.g., S. aureus), Rickettsiasp. (e.g., R. rickettsii), Treponema sp. (e.g., T. pallidum), Vibrio sp.(e.g., V. cholerae), Yersinia sp. (e.g., Y. enterocolitica, Y. pestis),Pneumocystis sp. (e.g., P. carinii), Aspergillis sp. (e.g., A.fumigatus, A. terreus, A. flavus), Candida sp. (e.g., C. albicans, C.krusei, C. tropicalis), Chlamidya sp., Schistosoma sp. (e.g., S.mansoni, S. japonicum, S. haematobium), Strongyloides stercoralis,Trichomonas sp., (e.g., T. vaginalis) and Tinea sp., (e.g., T. pedis).For any of these infections, a subject who has the infection, or issusceptible of developing the infection, e.g., by suspected exposure toan infectious agent, is treated by administering an effective amount ofa formula 1 compound to the subject. In these embodiments, the formula 1compounds can function by one or more mechanisms, including enhancinginnate immune responses, modulating, e.g., detectably increase ordecrease, the level or activity of one or more of the transcriptionfactors, enzymes or other biomolecules described herein, e.g., 1L-1α,TNFβ, IL-6, IL-8, IL-10, gro-α, IFN-γ, MCP-1, MIP-1α, MIP-1β, IP-10,GM-CSF, RANTES or their isotypes or homologs or cortisol. For example,molecules such as IL1α, TNFα, MIP-1α or MCP-1 are generally decreased ininfections where there is an overexpression of one or more of thesemolecules. Generally a decrease of one or more of these moleculesoccurs.

Fungal infections that can be treated, prevented or ameliorated thusinclude invasive aspergilliosis, allergic bronchopulmonaryaspergillosis, aspergilloma and chronic necrotizing aspergillosis.Bacterial infections that can be treated, prevented or ameliorated thusinclude infections by intracellular or extracellular gram positivebacteria, gram-negative bacteria, acid fast bacteria or by Mycoplasma.Other pathogens that are amenable to treatments according to the presentinvention are as described. See, e.g., J. B. Peter, editor, Use andInterpretation of Laboratory Tests in Infectious Disease, 5^(th)Edition, pages 1-309, 1998, Specialty Laboratories, Santa Monica,Calif., ISBN 1-889342-13-0.

In an exemplary embodiment, a subject such as a human that is known orsuspected of having been exposed to B. anthracis spores or cells istreated with a formula 1 compound. The subject may have overt symptomsof either cutaneous or pulmonary infection. The formula 1 compound isadministered at a dosage disclosed herein, e.g., about 0.05-10 mg/kg/dayor about 0.1-5 mg/kg/day by buccal delivery or by a parenteral routesuch as subcutaneous, intramuscular or intravenous injection, for about5-14 consecutive days. An oral dosage would be about 10-25 mg/kg/day ofa formula 1 compound for about 5-14 consecutive days. Dosing with theformula 1 compound will typically begin at about the time that theinfection is suspected or is diagnosed, or shortly thereafter, e.g.,within about 1-12 hours.

During or after treatment, the patient is optionally monitored and theamelioration of one or more symptoms or a slowed disease progression isobserved. Such symptoms can include one or more of a red-brown bump withswelling at the edges, blisters, formation of a black scab or eschar atthe site of skin infection, edema, respiratory difficulty and swellingof regional or local lymph nodes. Other symptoms of cutaneous anthraxthat can be ameliorated include fever, headache, muscle ache, nausea,and vomiting. In treating B. anthracis infections, the formula 1compounds will typically enhance innate immune responses, enhancehumoral immune responses, reduce TNFα, IL-1α or IL-1β levels or activityand/or enhance killing or phagocytosis of pathogen in the infectedsubject or the subject's immune cells, e.g., monocytes, neutrophils ormacrophages.

Treatment of a subject such as a human who is anticipated to potentiallycome in contact with a pathogen described herein, e.g., spores orvegetative B. anthracis cells, is accomplished by administering aformula 1 compound to the subject by, e.g., daily or intermittentdosing, beginning at about 1-14 days before an anticipated potentialexposure. The daily doses and routes of administration are essentiallyas described herein, e.g., for an adult human, about 0.05-5 mg/kg/day bybuccal delivery or by a parenteral route such as subcutaneous,intramuscular or intravenous injection for 1, 2, 3, 4, 5, 6, 7 or 8consecutive days or on alternate days over a 4, 6, 8, 10, 12 or 14 dayperiod in advance of a potential exposure.

For a pulmonary anthrax infection, amelioration of one or more of fever,bleeding and necrosis of lymph nodes near the lung, local chestinfection, shock, coma or death can occur. Infection of the brain andmeningoencephalitis may occur and is treated in a similar manner,although an increased dosage can be utilized, e.g., about 20-50mg/kg/day of the formula 1 compound is administered by a parenteral,e.g., intravenous, sublingual or buccal route. In any of these skin,pulmonary or gastrointestinal infections, the subject is also optionallytreated using one or more standard antibiotics and routes ofadministration, e.g., (1) 600,000 U i.m. bid for about 7-10 days ofprocaine penicillin G, optionally combined with about 500 mg/day ofstreptomycin, optionally administered q 8 h i.m., (2) 2 g/day oftetracycline for 5-10 days for an adult human, (3) oral erythromycinadministered at about 250 to 1000 mg/day in subdoses administered at 12or 8 hour intervals, (4) intravenous or oral ciprofloxacin for about5-30 days with daily doses optionally subdivided for administration 2 or3 times per day, e.g., about 250-500 mg of ciprofloxacin administeredtwice per day orally for a human adult, (5) doxycycline administered,e.g., at an induction dose of 100 mg twice on the first day and amaintenance dose of 50 mg twice per day for about 7-20 days for a humanadult, (6) a fluoroquinoline antibiotic such as levofloxacin,norfloxacin or oxofloxacin is administered according to standard dosingand administration protocols or (7) a corticosteroid for local orsystemic inflammation, e.g., of the lung, for a pulmonary anthraxinfection, which permits the corticosteroid to exert an antiinflammatoryactivity while avoiding at least some of the immune suppressiongenerally associated with corticosteroids.

The use of the formula 1 compounds will generally ameliorate theinflammation, sepsis or shock that can occur when antibiotics areadministered to subjects having a systemic or pulmonary B. anthracisinfection. A potential adverse effect of antibiotic use to treat asystemic or pulmonary B. anthracis infection is serious or potentiallylethal inflammation, sepsis and/or shock that results from release ofanthrax lethal toxin or factor or other inflammatory molecules on lysisof the bacteria. Release of bacterial lethal factor from lysed bacterialcells is associated with an intense inflammation, which is at leastpartially mediated by one or more inflammatory factors such as TNFα,IL-1β, IL-1α, IL-6, IL-8 or COX-2. The formula 1 compounds detectablyreduce the level and/or biological effects of such inflammatory factorsand can also detectably maintain or facilitate macrophage viability orone or more desired macrophage function(s) at the same time, e.g.,phagocytosis, killing of phagocytosed bacterial cells or debris orlimiting of reactive oxygen species generation by the macrophages.Similar considerations apply for the use of antibiotics to kill othertypes of bacteria, e.g., bacteria disclosed herein, that can releasepro-inflammatory molecules such as lipopolysaccharide, when anantibiotic kills or lyses bacterial cells.

Similarly, the formula 1 compounds can be used to treat, prevent orameliorate an infection by one or more gram-negative enteric bacteria.Such bacteria are commonly members of the Bartonella, Brucella,Campylobacter, Enterobacter, Escherichia, Francisella, Klebsiella,Morganella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia,Vibrio or Yersinia genera. For these infections, the formula 1 compoundis administered to a subject such as a human at a dosage disclosedherein, e.g., about 0.5-4 mg/kg/day by buccal delivery or by aparenteral route such as subcutaneous, intramuscular or intravenousinjection, for about 5-14 consecutive days. An oral dosage would beabout 10-25 mg/kg/day of a formula 1 compound for about 5-14 consecutivedays. Typically dosing with the formula 1 compound will begin at thetime that (or shortly thereafter, e.g., within about 1-12 hours) theinfection is suspected or is diagnosed. The patient is optionallymonitored and the amelioration of one or more symptoms or a sloweddisease progression is observed. The compounds can reduce the adverseeffects of bacterial lipopolysaccharide or endotoxin that is associatedwith these organisms. For example, the compounds are therapeuticallyuseful for infection by Yersinia pestis, which causes plague. Severalforms of plague can exist, i.e., bubonic, pneumonic, septicemic, orpestis minor. The compounds ameliorate one or more of the symptomsassociated with these infections. For example, in a bubonic plagueinfection, symptoms typically arise several days after exposure to Y.pestis, and can include a fever of up to 106° F., chills, rapid weakheartbeat, low blood pressure, lymph node swelling accompanied bytenderness, restlessness, confusion, uncoordinated movements, liver andspleen swelling. Symptoms associated with pneumonic plague include highfever, chills, rapid heartbeat, severe headache, coughing, blood-tingedsputum and rapid and labored breathing.

Treatment of a subject such as a human who is anticipated to potentiallycome in contact with cells of a gram negative enteric bacterium isaccomplished by administering a formula 1 compound to the subject by,e.g., daily or intermittent dosing, beginning at about 1-14 days beforean anticipated potential exposure. The daily doses and routes ofadministration are essentially as described herein, e.g., for an adulthuman, about 0.5-4 mg/kg/day by buccal delivery or by a parenteral routesuch as subcutaneous or intramuscular injection for 1, 2, 3, 4, 5, 6, 7or 8 consecutive days or on alternate days over a 4, 6, 8, 10, 12 or 14day period in advance of a potential exposure.

In Y. pestis infections, the subject is optionally treated using one ormore standard antibiotics and routes of administration, e.g., one ormore of (1) for septicemic or pneumonic infection, streptomycin at 30mg/kg/day is administered IM in 4 divided doses for about 7-10 days foran adult human, (2) tetracycline at 30 mg/kg/day is administered IV in 4divided doses for about 7-10 days for an adult human or (3) gentamycinor chloramphenicol according to standard doses and dosing routes.

In a subject having a V. cholerae infection, symptoms typically ariseseveral days after exposure to the pathogen, and can include a fever,chills, diarrhea, which can be serious or fatal if untreated, oliguria,muscle cramps and hypovolemia. In V. cholerae infections, the subject istreated with a formula 1 compound and optionally with one or morestandard therapies, e.g., one or more of (1) intravenous and/or oralreplacement of water, glucose and electrolytes, (2) tetracycline forabout 3-4 days at 500-1000 mg/day in 2, 3 or 4 optionally subdivideddoses for adult humans, (3) doxycycline at 300 mg/day for several days,(4) erythromycin, furazolidone norfloxacin, trimethoprim and/orsulfamethoxazole, according to standard dosages and routes ofadministration, e.g., oral or parenteral.

In any of these bacterial infections, the subject is optionally treatedwith a suitable or appropriate antibacterial agent(s). Such agentsinclude one, two or more antibacterial agents selected from anaminoglycoside, an amphenicol, an ansamycin, a β-lactam, a lincosamide,a macrolide, a peptide, a tetracycline, a 2,4-diaminopyrimidine, anitrofuran, a quinolone, a sulfonamide, a sulfone, cycloserine,mupirocin and tuberin, wherein the aminoglycoside optionally isdihdrostreptomycin, fortimicin(s), gentamicin, kanamycin, neomycin,neomycin undecylenate, spectinomycin, streptomycin, streptonicozid ortobramycin; the amphenicol optionally is azidamfenicol, chloramphenicol,chloramphenicol palmitate, chloramphenicol pantothenate, florfenicol orthiamphenicol; the ansamycin optionally is rifamide, rifampin orrifamycin; the β-lactam optionally is imipenem, cefactor, cefadroxil,cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, ceftibuten,ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephacetrile sodium,cephalexin, cephaloglycin, cephaloridie, cephalosporin, cephalothin,cephapirin sodium, amidinocillin, amdinocillin pivoxil, amoxicillin,apalcillin, carbenicillin, carfecillin sodium, carindacillin,floxicillin, a penicillin G, penicillin G potassium, penicillin Gprocaine, penicillin N, penicillin O, penicillin V or pivapicillin; thelincosamide optionally is clindamycin or lincomycin; the macrolideoptionally is azithromycin, clarithromycin or erythromycin; thepolypeptide optionally is amphomycin, bacitracin, a gramicidin,gramicidin S, mikamycin, polymyxin, polymyxin B-methanesulfonic acid, orzinc bacitracin; the tetracycline optionally is clomocycline,demeclocycline, doxycycline, oxytetracycline, or tetracycline; the2,4-diaminopyrimidine optionally is brodimoprim, tetroxoprim ortrimethoprim; the nitrofuran optionally is furaltadone, nifuradene,nifuratel, or nifurpirinol; the quinolone optionally is amifloxacin,cinoxacin, ciprofloxacin, difloxacin, enoxacin, fleroxacin, flumequine,nomefloxacin, miloxacin, nalidixic acid, norfloxacin, ofloxacin,oxolinic acid, pefloxacin, pipemidic acid, piromidic acid, rosoxacin,temafloxacin or tosufloxacin; the sulfonamide is optionally selectedfrom acetyl sulfamethoxypyrazine, azosulfamide, benzylsulfamide,chloramine-T, p-nitrosulfathiazole, succinylsulfathiazole orsulfathiazole; and the sulfone optionally is acedapsone, acetosulfonesodium, dapsone, solasulfone, succisulfone, sulfanilic acid, sulfoxonesodium or thiazolsulfone.

The formula 1 compounds are useful to detectably reduce the incidence,severity or progression of one or more symptoms or conditions associatedwith a viral, bacterial, fungal, yeast or parasite infection. Exemplarysymptoms and conditions associated with infections such as one or moreof those described above include one or more of sepsis, septicemia,fever, e.g., moderate to high fever, inflammation, pain, e.g., chestpain, muscle pain, joint pain, back pain or headache, chills, itching,rash, skin lesions, erythema, e.g., peripheral erythema,lymphadenopathy, e.g., local, regional or systemic lymphadenopathy,nausea, vomiting, cyanosis, shock, coma, necrosis, hemorrhage,encephalitis, meningoencephalitis, cramping, mild to severe diarrhea,cough, weakness, splenomegaly, anorexia and weight loss. Specificsymptoms that are associated with infections are known. See, e.g., TheMerck Manual, 17^(th) edition, M. H. Beers and R. Berkow editors, 1999,Merck Research Laboratories, Whitehouse Station, N.J., ISBN0911910-10-7, J. B. Peter, editor, Use and Interpretation of LaboratoryTests in Infectious Disease, 5^(th) Edition, pages 1-309, 1998,Specialty Laboratories, Santa Monica, Calif., ISBN 1-889342-13-0. Intreating any of these infections, the formula 1 compound will actthrough one or more mechanism, e.g., by decreasing TNF-α, IL-1α or IL-1βwhere, e.g., inflammation is a symptom, or by enhancing the activity ofcell mediated immune responses in clearing infected cells orextracellular infectious agents.

In any of the embodiments or treatment methods disclosed herein, one canoptionally administer an additional therapeutic treatment in conjunctionwith, i.e., before, during or after, administration of a formula 1compound(s) to a subject(s). For example, in subjects who have a viralor parasite infection and are in the course of administration of aformula 1 compound, other treatments can also be administered to thesubject, e.g., nucleoside analogs for viral infections or anantimalarial(s) agent such as one or more of artemisinin,dihydroartemisinin, a artemisinin analog (e.g., as disclosed in J. Hanet al., J. Nat. Products 64:2101-1205 2001 or G. A. Balint Pharmacol.Ther. 90:261-265 2001), dapsone, sulfadoxin, pyrimethamine, chloroquine,mefloquine, halofantrine, proguanil, proguanil hydrochloride,cycloguanil, chlorocycloguanil, atovaquone, quinine, berberine, and/orprimaquine for subjects having or subject to developing a malariainfection. Subjects suffering from or subject to developing a fungalinfection can optionally be treated with a formula 1 compound and anantifungal agent, e.g., an azole or a polyene such as ketoconazole,fluconazole, anidalfungin, amphotericin B or a liposomal formulationthat comprises an azole or polyene such as amphotericin B. Subjectssuffering from another condition such as an inflammation condition, anautoimmune condition or a cancer are optionally treated using one ormore additional treatments. Such additional treatments will typicallyinclude standard therapies for the subject's pathological condition(s),but they can also include experimental or other treatments. For example,one can coadminister vitamins (e.g., multivitamins, individualvitamins), antioxidants or other agents (e.g., vitamin E, allopurinol,folinic acid, carnitine, a C2-8 alkanoyl carnitine such as acetyl orpropionyl L-carnitine), nutritional supplements (e.g., liquid protein orcarbohydrate preparations) or other therapies as the patient's medicalcondition warrants or as the patient's doctor recommends. Any of theseadditional treatments can be coupled with the administration of any ofthe formula 1 compounds, e.g., BrEA, an ester, carbamate, carbonate oramino acid or peptide conjugate thereof, in any of the embodimentsdescribed herein.

Such additional therapeutic agents or therapeutic treatments areapparent to the skilled artisan. Such treatments are selected based onthe condition(s) to be treated, cross-reactivities of ingredients andpharmaco-properties of the combination. For example, when treating aviral infection(s), e.g., a retroviral infection, in a human or othersubject, the formula 1 compounds are combined with one or more reversetranscriptase inhibitors, protease inhibitors, antibiotics oranalgesics. Suitable formula 1 compounds that are combined with suchtherapeutic agents include those described, e.g., in the compoundgroups, embodiments or claims disclosed herein.

In some embodiments, the dosages and administration of an additionaltherapeutic agent or treatment will be used with a formula 1 compoundtreatment in essentially the same manner as usual for the agent ortreatment. In other embodiments, the use of the formula 1 compound iscoupled with an altered dosage or dosing protocol, either of which maybe appreciably increased or decreased. For example, in subjects such asadult humans that are infected with malaria parasites, the use of aformula 1 compound such as BrEA is optionally preceded by administrationof about 25-500 mg/day, e.g., about 50 mg/day, about 100 mg/day or about200 mg/day, of the antimalarial agent artemisinin for 1, 2, 3, 4, 5, 6,7 or more consecutive days followed one day after artemisinin ordihydroartemisinin dosing by administration of about 25-200 mg/day ofBrEA for 1, 2, 3, 4, 5, 6, 7 or more consecutive days. In relatedembodiments, the formula 1 compound and artemisinin are administered atpartly or completely overlapping times or the formula 1 compound isadministered before artemisinin or dihydroartemisinin is administered tothe infected subject. The daily dosage or the total dosage for a courseof therapy with other therapeutic agents such as cancer chemotherapyagents or antimicrobial agents (e.g., antiviral, antiparasite,antibacterial or antifungal agents as disclosed herein or in a citedreference) may be increased or decreased by at least about 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to standard dosagesfor such additional agents. The use of the formula 1 compounds withother therapeutic agents can reduce the toxicity of other therapeuticagents and/or enhance their potency or their therapeutic index (orselectivity), thereby allowing effective increased or decreased dosagesof the other therapeutic agent.

Exemplary antiviral agents suitable for use in the method includereverse transcriptase or polymerase inhibitors such as AZT (zidovudineor 3′-azido-3′-deoxythymidine), 3TC, D4T, ddI, ddC,2′,3′-dideoxynucleosides such as 2′,3′-didoxycytidine,2′,3′-dideoxyadenosine, 2′,3′-didoxyinosine, 2′,3′-didehydrothymidine,carbovir and acyclic nucleosides, e.g., acyclovir, ganciclovir.Exemplary protease inhibitors, fusion inhibitors or other antiviral orantiretroviral agents that may be used in a combination therapy with aformula 1 compound include lamivudine, indinavir, nelfinavir,amprenavir, ritonavir, crixivan, sequanavir, nevirapine, stavudine, aHIV fusion inhibitor, efavirenz, co-trimoxazole,N-(tert-butyl-dechydro-2)-2(R)-hydroxy-4-phenyl-3(S)-{N-2-quinolyl-carbonyl)-L-asparginyl!butyl}-(4a,S,8a,S)-isoquinoline-3(S)-carboxamide(Ro 31-8959), oxathiolan nucleoside analogues such ascis-1-(2-hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine orcis-1-(2-(hydroxymethyl)-1,3-oxathiolan-3-yl)-5-fluoro-cytosine,3′-deoxy-3′-fluoro-thymidine, 2′3′-dideoxy-5-ethynyl-3′-fluorouridine,5-chloro-2′,3′-dideoxy-3′-fluorouridine, ribavirin,9->4-hydroxy-2-(hydroxymethyl)but-1-yl-guanine (H2G), adefovirdipivoxil,9-[2-(R)-[[bis[[(isopropoxy-carbonyl)oxy]-methoxy]phosphinoyl]methoxy]propyl]adenine,(R)-9-[2-(phosphonomethoxy)-propyl]adenine, tenofivir disoproxil and itssalts (including the fumarate salt) and adefovir, TAT inhibitors such as7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one (R^(o5)-3335), or7-chloro-1,3-dihydro-5-(1H-pyrrol-2-yl)-3H-1,4-benzodiazepin-2-amine(Ro24-7429), renal excretion inhibitors such as probenecid, nucleosidetransport inhibitors such as dipyridamole; pentoxifylline,N-acetylcysteins, procysteine, α-trichosanthin, phosphonoformic acid, aswell as immunodulators or related agents such as interleukin II,granulocyte macrophage colony stimulating factors, erythropoetin,soluble CD4, tucaresol, 4-(2-formyl-3-hydroxy-phenoxymethyl)benzoic acidand oligonucleotides or nucleic acids that comprise one or moreunmethylated CpG sequences essentially as disclosed in, e.g., U.S. Pat.No. 6,194,388.

When treating other viral infections of the respiratory system, liver,blood, skin or other systems, e.g., human hepatitis C virus (“HCV”),human hepatitis B virus (“HBV”), HIV-1, HIV-2, an Orthopoxvirusinfection, a filovirus infection, a picornavirus infection or aninfluenza virus infection (e.g., human influenza A or B), a formula 1compound are optionally is used in conjunction with antivirals ortreatments for such viruses. Examples of such therapeutic agents ortreatments which are useful in these methods include carbovir,oxathiolan nucleoside analogs such ascis-1-(2-hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine orcis-1-(2-hydroxymethyl)-1,3-oxathiolan-5-yl-5-fluoro-cytosine,2′,3′-didoxy-5-ethynyl-3′-fluorouridine,5-chloro-2′,3′-didoxy-3′-fluorouridine,1-(6-D-arabinofuranosy)-5-propynyluracil, tenofovir, tenofovirdisoproxil, tenofovir disoproxil fumarate, bis(POC)-PMEA, bis(POM)-PMEA,bis(POC)-PMPA, bis(POM)-PMPA, acyclovir, HPMPC, amantadine, rimantadine,ribavirin, oseltamivir or compounds disclosed in U.S. Pat. Nos.5,763,483 (especially compounds recited in claims 1 and 2), 5,866,601and 6,043,230, mucolytics, expectorants, bronchodilators, antibiotics,antipyretics, analgesics or cytokines or interleukins that can augmentone or more aspects of a desired immune response, e.g., IL-1, IL-2,IL-3, IL-6, α-interferon, β-interferon, γ-interferon, G-CSF, GM-CSF,M-CSF or thrombopoietin. Such cytokines or interleukins can be used forany viral infection essentially according to known dosing methods anddosages, e.g., as disclosed herein or in the cited references.

In these treatment methods the infectious agent in the infected subject(or one susceptible to infection) may be sensitive or resistant to oneor more antimicrobial agents.

For example, bacteria causing an infection can be sensitive or resistantto antibiotics such as a penicillin (e.g., ampicillin, amoxicillin,ampicillan, carbenicillin, cloxacillin, cyclacillin, dicloxacillin,floxicillin, penicillin G, penicillin G calcium or penicillin N), acephalosporin (e.g., cefadroxil, cefamandole, or cefatrizine), a sulfadrug, a tetracycline, oxytetracycline, doxycycline, vancomycin,chloramphenicol, trimethoprim, erythromycin, rifampin, tobramycin,gentamycin, amikacin, a fluoroquinolone (e.g., ciprofloxacin,norfloxacin, ofloxacin, levofloxacin) or a quinoline. Infectingparasites can similarly be sensitive or resistant to antimicrobials,e.g., chloroquine resistant or sensitive Plasmodium parasites. Thus, insome embodiments, the subject's therapeutic regimen will optionallyinclude treatment of resistant or sensitive infectious agents with oneor more known antimicrobial agents. The antimicorbial can beadministered orally, by intravenous injection or drip or as otherwiseindicated by the subject's condition. Exemplary antimicrobial agents areas described, e.g., herein or in G. J. Galasso et al., editors,Antiviral Agents and Human Viral Diseases, 4^(th) edition, pages 1-833,1997, Lippincott-Raven, Philadelphia, Pa., ISBN 0-397-51709-2, U.S. Pat.No. 4,753,925, or in any reference cited herein. Invention embodimentsinclude treating a subject who has a bacterial or parasite infection asdescribed with a formula 1 compound as described herein and a anantibacterial agent(s) or an antiparasitic agent(s) as described hereinor in the cited references.

The antiviral or antimicrobial agents or treatments in combinationtherapies with a formula 1 compound will be or are used essentiallyaccording to new or to known dosing and administration methods for thoseagents or treatments. Their use may precede, overlap or be coincident intime with or follow a treatment protocol with a formula 1 compound. Insome embodiments, the other therapeutic agents or treatments willoverlap and will thus be administered on one or more of the same days onwhich a formula 1 compound is administered to a subject having a viralinfection, or subject to a viral infection. In other embodiments, theother therapeutic agents or treatments will be administered to such asubject within about 1 day to about 180 days before or after a treatmentprotocol or a dosing period with a formula 1 compound begins or ends. Inexemplary embodiments, the other suitable treatment or agent isadministered within 1 day, 2 days, 3 days, 4 days, about 7 days, about14 days, about 28 days or about 60 days before or after a treatmentprotocol or a dosing period with a formula 1 compound begins or ends.

Although the forgoing combination therapies have been described in thecontext of viral or other infections, the protocols and methods thatemploy a formula 1 compound can be used in conjunction with any suitablenew or known therapeutic agent(s) or treatment protocol(s) for other anyother clinical condition described herein. Exemplary conditions includeone or more of a non-viral pathogen infection(s), a cancer(s), aprecancer(s), an inflammation condition(s), an autoimmune condition(s),an immunosuppression condition(s), a neurological disorder(s), acardiovascular disorder(s), a neurological disorder(s), diabetes,obesity, wasting, anorexia, anorexia nervosa, a cancer chemotherapy(ies)side-effect(s), a side-effect(s) of a chemotherapy(ies) or a radiationtherapy(ies) of any other clinical condition disclosed herein or in thecited references, or the like. Thus, invention embodiments include theuse of a formula 1 compound before, during or after a treatment thatuses another suitable therapeutic agent(s) or therapeutic treatment(s)for any of the diseases or conditions disclosed herein, any of whichdiseases or conditions may be acute, chronic, severe, mild, moderate,stable or progressing.

Examples of such agents or treatments include the use of one or moreadrenergic agents, adrenocortical suppressants, aldosterone antagonists,anabolics, analeptics, analgesics, anesthesia, anthelmintics, antiacneagents, anti-adrenergics, anti-allergics, anti-amebics, anti-androgens,antianginals, anti-anxiety agents, anti-arthritics, anti-asthmaticagents, anti-atherosclerotic agents, antibacterials, anticholinergics,anticoagulants, anticonvulsants, antidepressants, antidiabetics,antidiarrheals, antidiuretics, anti-emetics, anti-epileptics,anti-estrogens, antifibrinolytics, antifungals, antihistamines,antihyperlipidemia agents, antihyperlipoproteinemic agents,antihypertensive agents, antihypotensives, anti-infectives,anti-inflammatory agents such as entanercept (a dimeric fusioncomprising a portion of the human TNF receptor linked to the Fc portionof human IgG1 containing the C_(H)2 and C_(H)3 domain and hinge regionsof IgG1) or a COX-2 inhibitor such as celexicob(4-5[-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazole-1-yl]benzenesulfonamide)or rofecoxib (4-[4-methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone),antimalarial agents, antimicrobials, antimigraine agents, antimycoticagents, antinausea agents, antineoplastic agents, antiparasitics,antiparkinsonian agents, antiproliferatives, antiprostatic hypertrophyagents, antiprotozoals, antipruritics, antipsychotics, antirheumatics,antischistosomals (e.g., praziquantel, artemisinin), blood glucoseregulators, bone resorption inhibitors, bronchodilators, cardiacdepressants, cardioprotectants, choleretics, depressants, diuretics,dopaminergic agents, enzyme inhibitors, free oxygen radical scavengers,glucocorticoids, peptide hormones, steroid hormones,hypocholesterolemics, hypoglycemics, hypolipidemics, hypotensives,immunomodulators, liver disorder treatments, mucosal protective agents,nasal decongestants, neuromuscular blocking agents, plasminogenactivators, platelet activating factor antagonists, platelet aggregationinhibitors, post-stroke and post-head trauma treatments, progestins,psychotropics, radioactive agents, relaxants, sclerosing agents,sedatives, sedative-hypnotics, selective adenosine A1 antagonists,serotonin antagonists, serotonin inhibitors, serotonin receptorantagonists, thyroid inhibitors, thyromimetics, tranquilizers,vasoconstrictors, vasodilators, wound healing agents, xanthine oxidaseinhibitors or a treatment(s) or therapeutic agent(s) for amyotrophiclateral sclerosis, ischemia, e.g., cereberal ischemia, cardiac ischemiaor cardiovascular ischemia, or unstable angina. The selection and use ofthese agents for a particular subject will typically use dosing methods,dosages and routes of administration essentially according to knownmethods, dosages and routes of administration. Such methods, dosages androutes of administration are described in detail at, e.g., Textbook ofAutoimmune Diseases, R. G. Lahita, editor, Lippincott Williams & Wikins,Philadelphia, Pa., 2000, ISBN 0-7817-1505-9, pages 81-851, Holland•FreiCancer Medicine ^(e.)5, 5^(th) edition, R. C. Bast et al., editors,2000, ISBN 1-55009-113-1, pages 168-2453, B. C. Becker Inc. Hamilton,Ontario, Canada, Hematology, Basic Principles and Practice, 3^(rd)edition, R. Hoffman, et al., editors, 2000, ISBN 0-443-77954-4, pages115-2519, Churchill Livingstone, Philadelphia, Pa., Rheumatology, 2^(nd)edition, J. H. Klippel et al., editors, 1998, ISBN 0-7234-2405-5, volume1, sections 1-5 and volume 2, sections 6-8, Mosby International, London,UK, Alzheimer's Disease and Related Disorders: Etiology, Pathogenesisand Therapeutics, K. Iqbal, et al., editors, 1999, ISBN 0-471986386,John Wiley & Son Ltd, and Cardiovascular Medicine, E. J. Topol, editor,Lippincott Williams & Wikins, Philadelphia, Pa., 1998, ISBN 0781716810.

In some infections, the formula 1 compound(s) effects an improvement ofone or more of the symptoms associated with the infection or a symptomthereof. For example, treatment of subjects who are immune suppressed,e.g., from a retrovirus infection, cancer chemotherapy or other cause,generally show improvement of one or more associated symptoms, such asweight loss, fever, anemia, pain, fatigue or reduced infection symptomsthat are associated with a secondary infection(s), e.g., HSV-1, HSV-2,papilloma, human cytomegalovirus (“CMV”), Pneumocystis (e.g., P.carinii) or Candida (C. albicans, C. krusei, C. tropicalis) infections.

In some embodiments, the formula 1 compound(s) is administered as anonaqueous liquid formulation as described herein or the formula 1compound(s) is administered according to any of the intermittent dosingprotocols described herein using a solid or liquid formulation(s). Inthe case of a subject who has a retroviral infection, e.g., a human withan HIV infection, with symptoms that include one or more of, arelatively low CD4 count (e.g., about 10-200, or about 20-100 or about20-50), one or more additional pathogen infections (HSV-1, HSV-2, HHV-6,HHV-8, CMV, HCV, a HPV, P. carinii or Candida infection) and one or moreof anemia, fatigue, Kaposi's sarcoma, fever or involuntary weight loss(at least about 5% of body weight), administration of about 0.1 to about10 mg/kg/day (usually about 0.4 to about 5 mg/kg/day) of a formula 1compound(s) to the subject typically results in noticeable improvementof one or more of the symptoms within about 1-4 weeks. In otherembodiments, the formula 1 compound(s) is administered to a subject whohas a condition that appears to be associated with a viral infection,e.g., pneumonia or retinitis associated with CMV, nasopharyngealcarcinoma or oral hairy leukoplakia associated with Epstein-Barr virus,progressive pancephalitis or diabetes associated with Rubella virus oraplastic crisis in hemolytic anemia associated with Parvovirus 19.

One or more intermittent dosing protocols disclosed herein or one ormore of the liquid non-aqueous formulations described herein can beapplied by routine experimentation to any of the uses or applicationsdescribed herein. For a formula 1 compound(s) that is a new compound perse, the compound(s) can be administered to a subject according to aninvention intermittent dosing protocol(s) or by other protocols, e.g.,continuous daily dosing of a single dose or two or more subdoses perday. In addition any of the formula 1 compounds, e.g., one or moreformula 1 compounds that are new per se, can be present in any solid orliquid formulation described herein. These formulations and dosingprotocols can be applied by routine methods to any of the uses orapplications described herein.

Antibodies, vaccines and vaccine adjuvants. The formula 1 compoundsdisclosed herein may also be used as vaccine adjuvants with immunogensor components of immunogenic compositions to prepare antibodies capableof binding specifically to the formula 1 compounds, their metabolicproducts which retain immunologically recognized epitopes (sites ofantibody binding) or prepare antibodies that bind to antigens that canbe used for vaccination against, e.g., infectious agents or malignantcells. The immunogenic compositions therefore are useful asintermediates in the preparation of antibodies that bind to formula 1compounds for use, e.g., in diagnostic, quality control, or the like,methods or in assays for the compounds or their novel metabolicproducts. In addition, the compounds are useful for raising antibodiesagainst otherwise non-immunogenic polypeptides, in that the compoundsmay serve as haptenic sites stimulating an immune response against thepolypeptide.

The hydrolysis products or metabolites of formula 1 compounds includeproducts of the hydrolysis of the protected acidic, basic or otherreactive groups that variable groups, e.g., R¹-R⁹, optionally comprise.In some embodiments, acidic or basic amides comprising immunogenicpolypeptides such as albumin (e.g., human or mammalian), keyhole limpethemocyamin and any other peptide described herein are used asimmunogens. The metabolites of formula 1 compounds may retain asubstantial degree of immunological cross reactivity with theunmetabolized parent compounds. Thus, in some embodiments, theantibodies will be capable of binding to the metabolites of the parentformula 1 compound without binding to the parent compound itself. Inother embodiments, the antibodies, will be capable of binding to theparent compounds only, while in other cases the antibodies will becapable of binding to either of these. Some of the antibodies will notsubstantially cross-react with naturally occurring materials or epitopesthat are present in the subject. Substantial cross-reactivity isreactivity under specific assay conditions for specific analytessufficient to interfere with the assay results.

The immunogens of this invention may comprise a formula 1 compound thathas 1 or more epitopes in association with another immunogenicsubstance. Within the context of the invention such association meanscovalent bonding to form an immunogenic conjugate (when applicable) or amixture of non-covalently bonded materials, or a combination of theabove. Immunogenic substances include adjuvants such as Freund'sadjuvant, immunogenic proteins such as viral, bacterial, yeast, plantand animal polypeptides, including keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin or soybean trypsin inhibitor, andimmunogenic polysaccharides. Typically, the formula 1 compound havingone, two or more epitopes is covalently conjugated to an immunogenicpolypeptide or polysaccharide by the use of a polyfunctional (ordinarilybifunctional) cross-linking agent. Methods for the manufacture ofimmunogens that comprise one or more haptens are conventional per se.Any suitable known method for conjugating haptens to immunogenicpolypeptides or the like are used here, taking into account thefunctional groups on the precursors or hydrolytic products which areavailable for cross-linking and the likelihood of producing antibodiesspecific to the epitope in question as opposed to the immunogenicsubstance.

Typically a polypeptide, polysaccharide or other suitable immunogenicmoiety is conjugated to a site on a formula 1 compound in a locationthat is distant from the epitope on the formula 1 compound to berecognized.

The conjugates are prepared in conventional fashion. For example, thecross-linking agents N-hydroxysuccinimide, succinic anhydride or C₂₋₈alkyl-N═C═N—C₂₋₈ alkyl are useful in preparing the conjugates. Theconjugates comprise a formula 1 compound that is attached by a bond or alinking group of 1-100, typically, 1-25, more typically about 1-10carbon atoms to the immunogenic substance. The conjugates are separatedfrom starting materials and by-products using chromatography or thelike, and then are optionally sterile filtered, or otherwise sterilized,or are optionally vialed for storage. Synthetic methods to preparehapten-carrier immunogens have been described, see e.g., G. T.Hermanson, Bioconjugate Techniques Academic Press, 1996, pages 419-493.

The formula 1 compounds are cross-linked for example through any one ormore of the following groups: a hydroxyl group, a thiol group, acarboxyl group, a carbon atom, or an amine group. Included within suchcompounds are amides of polypeptides where the polypeptide serves as anabove-described protecting group.

Animals or mammals are typically immunized once, twice or more timesagainst the immunogenic conjugates that comprise a formula 1 compound ortheir derivatives and polyclonal antisera or monoclonal antibodiesprepared in conventional fashion. In some embodiments, about 0.0001mg/kg to about 1 mg/kg, e.g., about 0.001 or about 0.01 or about 0.1mg/kg, of immunogenic conjugate or derivative is used on one, two, threeor more occasions to immunize the subject as described herein. Theimmunogenic conjugates are administered, orally, topically orparenterally as described herein, e.g., by i.m. or s.c. injection.Methods to prepare antibodies, including methods to obtain antibodiesthat bind to steroids have been described, see, e.g., R. O, Neri et al.,Endocrinology 74:593-598 1964, M. Ferin et al., Endocrinology85:1070-1078 1969, J. Vaitukaitis et al., J. Clin. Endocr. Metab.33:988-991 1971 and M. Ferin et al., Endocrinology 94:765-775 1974. Suchmethods can be used essentially as described to prepare antibodies ormonoclonal antibodies that bind to a formula 1 compound. Inventionembodiments include serum or other preparations that comprise anypolyclonal or monoclonal antibodies that bind to a formula 1compound(s), methods to make them and compounds or compositions that areused in conducting these methods.

In other embodiments the formula 1 compounds are used as adjuvants toenhance a subject's immune response to antigens such as proteins,peptides, polysaccharides, glycoproteins or killed or attenuated virusesor cell preparations. In these methods, an effective amount of theformula 1 compound is administered at about the same time that theantigen is delivered to the subject, e.g., within about 1, 2, 3, 4, 5,6, or 7 days of when the antigen is administered to the subject. In someembodiments, the formula 1 compound is administered 1, 2, 3, 4 or moretimes (usually once or twice per day) at 1, 2, 3 or 4 days before orafter the antigen is administered to the subject. In other embodiments,the formula 1 compound is administered on the same day that the antigenis administered to the subject, e.g., within about 1-4 hours. Suchimmunization methods may be repeated once, twice or more as needed. Theformula 1 compound can be administered to the subject using any of theformulations or delivery methods described herein or in the referencescited herein. Subjects suitable for these vaccinations include young andelderly mammals, including humans, e.g., humans about 3-36 months of ageor older and humans about 60, 65, 70, 75 years of age or older. Theamount of antigen used can be about 0.01 μg/kg to about 20 mg/kg,typically about 1-100 μg/kg. Dosages of the formula 1 compound used inthese vaccinations is essentially as described herein, e.g., about 10 mgto about 1000 mg of a formula 1 compound is used per day on days when itis administered as part of the vaccination method.

Related embodiments include compositions or formulations that comprise aformula 1 compound, an antigen(s) or antigen(s) preparation andoptionally one or more excipients. The antigen is essentially asdisclosed herein or in a cited reference. Antigen preparations maycomprise one or more of (1) lethally or sublethally radiated cells orpathogens, (2) disrupted cells or viruses or such as attenuated viruses,(3) a nucleic acid or DNA vaccine, (4) an antigenic protein,glycoprotein, polysaccharide or a fragment or derivative of any of thesemolecules, (5) chemically treated cells or pathogens, e.g., formalin ordetergent treated cells, viruses or cell or virus extracts and (6)genetically engineered viral or bacterial vectors that express one ormore antigens or antigen fragments, e.g., canarypox virus containing HIVor other pathogen sequences or other animal viruses carrying HIV orother pathogen sequences. Pathogens include prions or the etiologicagents of, e.g., Creutzfelt-Jacob disease, bovine spongiformencephalopathy and scrapie in sheep, goats or mice. Where cells ordisrupted are present in an antigen preparation, they may by geneticallymodified, e.g., to express one or more antigens or epitopes againstwhich an immune response is desired. Such cells may also be geneticallymodified to optionally express one or more factors, e.g., an interleukinor a cytokine, such as one described herein, to enhance the desiredimmune response. As used here, an antigen means a moiety that can beused to elicit an immune response when it is administered to a subject.In some embodiments, the antigen is foreign to the subject. For foreignantigens, the subject to be vaccinated may not encode or express theantigen, while the antigen is usually part of or expressed by a pathogenor by a subject or mammal of a different species. In other embodiments,antigens are endogenous or non-foreign to the subject, e.g., they areusually encoded or expressed by the subject or another subject of thesame species. Endogenous antigens are suitable for use in, e.g., tumorvaccination methods.

Exemplary tumors from which a suitable antigen(s) may be obtained are asdescribed herein or in the cited references. A DNA vaccine as used heretypically comprises a nucleic acid, usually DNA, that encodes one ormore antigens or epitopes that a pathogen, e.g., a parasite, fungus,virus or bacterium, or a tumor encodes or can express. Tumor antigensthat are suitable for use in vaccination methods that employ a formula 1compound include tumor-associated antigens and tumor-specific antigens.These molecules typically comprise one or more protein, glycoprotein,carbohydrate or glycolipid. Vaccinations that employ a tumor antigen(s)may comprise autologous tumor cells or allogenic tumor cells, which areoptionally disrupted and optionally used with a non-formula 1 adjuvant,such as bacillus Calmette-Guerin (BCG), purified protein derivative,Freund's complete adjuvant, Corynebacterium parvum, Mycobacteriumvaccae, oligonucleotides that consist of or comprise an unmethylated CpGdimer or an alum precipitate. In some embodiments, tumor cells treatedwith neuraminidase comprise all or part of the tumor antigen source. Thenon-formula 1 adjuvants are also optionally used in any of thevaccination methods disclosed herein. As used here, tumor associatedantigens, e.g., the carcinoembryonic antigen, α-fetoprotein or theprostate specific antigen, are molecules that are often associated withor detectably expressed by premalignant or malignant cells or cellpopulations and also with some normal tissues during at least part ofthe subject's life cycle.

Tumor-specific antigens, e.g., the R24c mutation of the cyclin dependentkinase-4 protein or certain sialylated glycoconjugates such as proteincontaining N-glycolyl neuraminic acid that is found in some human tumors(e.g., colon cancers, liver cancer, lymphoma), are molecules whoseexpression is restricted to pretumors or tumors and they are notexpressed in normal adult or fetal tissue of a subject or a species to asignificant extent. Vaccination with one or more tumor antigens and aformula 1 compound may be administered to a subject who has a cancer ora precancer, or to a subject who is considered potentially susceptibleto developing such a condition. The tumor antigens are optionallycombined with protein or other non-formula 1 adjuvants, e.g.,keyhole-limpet hemocyanin or an immunoglobulin from a different species,which may be covalently bonded to the tumor antigen(s).

Suitable natural and synthetic nucleic acid sequences, cells, attenuatedpathogens or infectious agents such as attenuated viruses, and protein,glycoprotein, polysaccharide, oligosaccharide or peptide antigensderived various infectious agents and tumors have been described, e.g.,U.S. Pat. Nos. 4,053,585, 4,081,334, 4,115,543, 4,503,036, 4,466,917,4,508,708, 4,601,903, 4,632,830, 4,683,200, 4,727,136, 4,735,799,4,784,850, 4,784,941, 4,803,164, 4,831,121, 4,831,126, 4,853,333,4,863,200, 4,857,452, 4,916,055, 4,939,240, 4,960,716, 4,963,484,5,013,661, 5,032,397, 5,075,218, 5,077,220, 5,093,118, 5,011,920,5,110,588, 5,112,749, 5,126,264, 5,134,075, 5,162,226, 5,185,432,5,198,535, 5,231,168, 5,225,193, 5,283,321, 5,302,386, 5,328,835,5,378,814, 5,393,532, 5,395,614, 5,456,911, 5,474,900, 5,478,556,5,348,887, 5,455,332, 5,489,525, 5,527,891, 5,541,292, 5,582,831,5,591,596, 5,609,872, 5,614,194, 5,639,621, 5,639,863, 5,641,492,5,643,567, 5,654,136, 5,679,342, 5,688,657, 5,688,658, 5,705,341,5,712,118, 5,723,130, 5,747,028, 5,756,101, 5,780,591, 5,798,445,5,814,617, 5,824,316, 5,824,777, 5,837,830, 5,843,451, 5,844,075,5,849,306, 5,849,476, 5,858,685, 5,866,679, 5,871,936, 5,874,060,5,895,285, 5,895,651, 5,916,571, 5,916,754, 5,916,879, 5,932,412,5,935,818, 5,942,235, 5,948,410, 5,948,412, 5,961,985, 5,968,514,5,985,571, 5,993,813, 5,993,828, 5,994,523, 6,013,765, 6,013,779,6,017,527, 6,020,478, 6,024,961, 6,025,191, 6,025,474, 6,030,624,6,030,797, 6,045,802, 6,056,963, 6,060,280, 6,083,703, 6,083,683,6,087,441, 6,093,540, 6,096,320, 6,100,049, 6,100,088, 6,100,241,6,100,444, 6,110,468, 6,110,724, 6,110,898, 6,120,770, 6,113,917,6,120,770, 6,127,116, 6,127,333, 6,130,082, 6,207,170, PCT publicationNos. WO 0025820, WO 0050645, WO 0050897, WO 0050900, WO 0052165, WO0057904, WO 0057906, WO 0097907, WO 00/58438 and R. C. Bast et al.,editors, Holland•Frei Cancer Medicine ^(e.)5, 5^(th) edition, 2000,pages 800-814, B. C. Becker Inc. Hamilton, Ontario. Antigens suitablefor use with the formula 1 compounds include the molecules disclosed inany of these references or antigenic fragments thereof. Such antigenicfragments will typically retain at least about 20% of the antigenicityof the unmodified antigen. Thus, these fragments will retain at leastabout 20% of the native antigen's capacity to generate an antibodyresponse or to generate a T cell response against the unmodified antigenor the like.

Other suitable antigens include STn, sialyl Tn-KLH, carbohydrateconjugates, carcinogenic embryonic antigen, MAGE-1, MUC-1, HER-2/neu,prostate specific antigen, p53, T/Tn, bacterial flagella antigens orcapsular polysaccharide antigens (e.g., Staphylococcus aureus capsularpolysaccharide antigens) and antigenic fragments or antigenic syntheticderivatives of any of these molecules. See, e.g., L. A. Holmberg et al.,Bone Marrow Transplant. 2000 25:1233-1241, J. W. Hadden, Int. J.Immunopharmacology 1999 21:79-101, G. Ragupathi et al., Glycoconj. J.1998 15:217-221, A. I. Fattom et al., Infect. Immun. 1998 66:4588-4592,U.S. Pat. Nos. 5,770,208, 5,866,140 and 6,194,161 and citationselsewhere herein, including the preceding paragraph.

An antigenic protein, peptide or glycoprotein can be identified bystandard methods, e.g., protein or nucleic acid sequencing, for any ofthe infectious agents or tumors that are described herein or in thecited references. Thus, in some embodiments, an effective amount of aformula 1 compound and an antigen are administered to a subject, ordelivered to the subject's tissues, to stimulate an immune responseagainst the antigen. The antigen may comprise one, two or more antigenicepitopes, which may come from one, two or more genes. In someembodiments, the subject is optionally monitored to follow or determinethe immune, dendritic cell, B cell, T cell, antibody or cytokineresponse, such as one disclosed herein, e.g., modulation or increase inγIFN, IL-2 or IL-12 levels or measurement of the production of one ormore immunoglobulin types or subtypes. The subject may also be monitoredby in vitro cell assays, e.g., for activation of T cells or subsets of Tcells or other relevant white blood cell types. Such assays includemeasuring T cell activation using chromium release assays, or mixedlymphocyte assays. The subject is optionally treated with one or moreadditional booster vaccinations, when this is called for under thecircumstances.

Nucleic acid or DNA vaccines as used here will typically comprise anucleic acid comprising an expressible region that encodes one, two ormore suitable antigens or epitopes, e.g., all or an antigenic portion ofa viral, bacterial, fungal or parasite protein or glycoprotein. Theexpressible region will usually comprise a transcription promoter andoptionally other control sequences that are operatively linked to theantigen coding region where the promoter and control sequences aretranscriptionally active in the intended subject or tissue. Suitablecontrol sequences include enhancers, recognition sequences fortranscription factors and termination sequences. Such expression vectorsmay optionally comprise one, two or more expressible genes or genefragments, which may each comprise their attendant operatively linkedexpression sequences. Suitable methods and expression vectors to delivernucleic acids for vaccine purposes have been described, e.g., U.S. Pat.Nos. 5,223,263, 5,580,859, 5,703,055, 5,846,946 and 5,910,488.

Thus, in some embodiments, an effective amount of a formula 1 compoundand an antigen are administered to a subject to stimulate an immuneresponse against the antigen, wherein the antigen is encoded by asuitable expression construct. Exemplary antigens that are suitable inthese methods are, e.g., essentially as described in any reference citedherein or as is apparent to the skilled artisan. In some embodiments,the antigen is encoded by a parasite such as a Plasmodium or aTrypanosome species such as one described herein. The formula 1 compoundcan be administered before, essentially simultaneously with or afteradministration of the antigen, as noted above. The dosages of theformula 1 compound are essentially as described for the other conditionsdescribed herein.

Vaccinations that utilize a formula 1 compound and an antigen(s) aregenerally suitable for eliciting or enhancing desired immune responsesin conjunction with exposure of a subject to an antigen(s), compared tovaccination without the compound. Antigen specific humoral antibodyresponses or antigen specific T cell responses may be enhanced orelicited. Typically vaccination using a formula 1 compound and asuitable antigen is conducted to prevent a potential infection or toreduce the severity of a future infection. However, in some cases thevaccination is conducted in a subject that has an infection such as achronic or a latent infection such as a parasite or a retrovirus orherpesvirus infection, which may be latent or in relapse. In other casesthe subject may have a cancer or precancer. Thus, the subject may beexposed to, or contain, one or more of the antigens that are used in oneof these vaccination procedures. Such vaccinations are included withinthe scope of the invention.

In related embodiments, the formula 1 compounds are useful to facilitatepreparation of hybridoma clones that express monoclonal antibodies. Inthese methods, a suitable amount of a formula 1 compound, e.g., about100 μg to about 2 mg for a small mammal, is administered to a subject,e.g., a mouse, to enhance the immune response to the desired antigen,which is also administered to the subject. After antigen challenge,suitable cells are recovered from the subject, e.g., anti-antigenimmunoglobulin expressing HPRT⁺ spleen cells from a mouse. These cellsare then fused with suitable immortal cells (e.g., mouse melanoma cells)using, e.g., PEG or Sendai virus, and selected in suitable selectiongrowth medium, e.g., tissue culture medium that contains hypoxanthine,aminopterin and thymidine, to obtain a group or panel of hybridomas thatexpress anti-antigen monoclonal antibodies. The hybridoma panel is usedto generate individual clones, which are optionally screened todetermine the antibody specificity and antigen binding properties. Aboutone, 100, 1000, 10,000, 100,000 or more individual clones are screenedby standard methods. The monoclonal antibodies may be from any suitablesource, e.g., murine, human, human-murine hybrid or the like. Methods toobtain human, human-murine hybrid or related monoclonal antibodies havebeen described, e.g., U.S. Pat. Nos. 5,562,903, 5,461,760, 5,705,154,5,854,400, 5,858,728, 5,874,082, 5,874,540, 5,877,293, 5,882,644,5,886,152, 5,889,157, 5,891,996, 5,916,771, 5,939,598, 5,985,615,5,998,209, 6,013,256, 6,075,181, 6,901,001, 6,114,143, 6,114,598,6,117,980. The formula 1 compounds can be used in any of the methodsdisclosed in these references to facilitate generation or recovery ofhybridoma panels and clones that express monoclonal antibodies.

An aspect of these methods comprise a product, i.e., a hybridoma panelor a hybridoma clone, that is obtained by the process of contacting asubject (such as a mouse) with (1) a suitable amount of a formula 1compound and (2) a suitable amount of an antigen, allowing sufficienttime to generate an immune response in the subject against the antigenand then fusing suitable anti-antigen immunoglobulin producing cellsfrom the subject, e.g., the subject's spleen cells, with a suitableimmortal cell line (e.g., a HPRT⁺ mouse myeloma). The antigen orimmunogen is as described above, e.g., a suitable protein, proteinfragment or glycoprotein such as an interleukin, cytokine or antigenfrom an infectious agent. In these methods, a mouse is typically thesubject, but other mammals, e.g., humans or other rodents, are alsosuitable according to known methods.

The amount of antigen for immunization used in preparing monoclonalantibodies in a small mammal will typically be about 1 μg to about 100μg, e.g., about 2 μg, 5 μg, 10 μg or 50 μg of antigen. The antigens areessentially as described in the vaccination methods described above,e.g., disrupted cell, a protein or glycoprotein, which is optionallycombined with a suitable amount of an adjuvant such as Freund's completeadjuvant, alum precipitate, a bacterial lipopolysaccharide or BCG. Theformula 1 compound is typically parenterally administered, e.g.,subcutaneous or intraperitoneal, within about 2-4 days (e.g., about 1,2, 3, 4, 5 or 6 days before or after antigen challenge) of the time thatthe subject is challenged with antigen. In some cases, the antigen andthe formula 1 compound is administered at the same time or at about thesame time, e.g., within about 5 minutes to about 1 hour. The formula 1compound may be administered on one, two, three or more occasions in theprocess, e.g., a formula 1 compound is administered once per day on oneor more of the four days before antigen challenge and then it isoptionally administered again on the same day as antigen challenge andthen optionally administered daily at one, two, three or more days afterantigen challenge.

Related embodiments include a method comprising administering to asubject (e.g., a mammal such as a human or a primate), or delivering tothe subject's tissues, an effective amount of a formula 1 compound and aspecific antigen. Immune responses that are enhanced include a mucosalimmune response to an antigen such as a protein, peptide,polysaccharide, microorganism, tumor cell extract or lethally radiatedtumor or pathogen cells (e.g., antigens or cells from melanoma, renalcell carcinoma, breast cancer, prostate cancer, benign prostatichyperplasia, virus or bacteria, or other tumor or pathogen as disclosedherein). Aspects of these embodiments include enhancement of thesubject's immune response when an antigen or immunogen is administeredintranasally or orally. In these aspects, the formula 1 compound isadministered about simultaneously with the antigen or within about 3hours to about 6 days of antigen administration. The use of immunemodulating agents to enhance immune responses to a vaccine has beendescribed, e.g., U.S. Pat. No. 5,518,725.

Other uses for the formula 1 compound(s) include administering thecompound(s) to a subject who suffers from a pathological condition(s).The treatment may treat or ameliorate the source of the condition(s)and/or symptoms associated with the pathological condition(s) such asinfection with a pathogen(s) (viruses, bacteria, fungi), a malignancy,unwanted immune response, i.e., an immune response that causes pathologyand/or symptoms, e.g., autoimmune conditions or allergy or conditionssuch as hypoproliferation conditions, e.g., normal or impaired tissuegrowth, or wound healing or burn healing, or in immunosuppressionconditions, e.g., conditions characterized by an absence of a desiredresponse and/or an inadequate degree of a desired response.

As noted in the foregoing applications where a formula 1 compound and anantigen are administered to a subject to enhance the subject's immuneresponse, the antigen may be obtained from any suitable source. Theantigen will generally be capable of eliciting an immune responseagainst the original pathogen or cell. Desirable immune responsesobtained from vaccination of a subject with an antigen and a formula 1compound include one or more of an enhanced antibody response, anenhanced antigen specific CD4⁺ T cell response or an enhanced cytotoxicT cell response to pathogen infected cells, extracellular pathogen or totumor cells.

Enhanced antibody responses include detectable enhancement of antibodytiter or a shift in the antibody response from a Th2 biased response toan increased Th1 biased component of the response. In such antibodyshifts, the Th1 and Th2 character of the response is determined by knownmethods. For example, a relatively low ratio of IgG1 (or the analogousantibody subclass in humans and other subjects) to IgG2a (or theanalogous antibody subclass in humans and other subjects), e.g., about6:1 to about 12:1, that is generated after exposure of a subject (amouse for the IgG1 and IgG2a subclasses) to an antigen indicates a Th1biased antibody response. Conversely a higher ratio, e.g., about 20:1 toabout 30:1 indicates a Th1 biased antibody response. Generation ofantigen-specific IgG1 generation involves T-helper type 2 (Th2) cells,and for IgG2a, T-helper type 1 (Th1) cells. The formula 1 compounds candetectably increase the Th1 character of an antibody response to anantigen or they can increase the magnitude of both the Th1 and Th2response.

Exemplary antigen sources include pathogens, cells or their individualproteins, peptides, glycoproteins or polysaccharides or antigenicfragments of any of these molecules. The antigenic material is recoveredfrom suitably treated pathogens or cells and administered to a subject,or it can be recovered using recombinant means to obtain a purified orpartially purified antigen source.

Exemplary pathogens or cells that are suitable sources for antigens or agene(s) that encode suitable antigens include influenza viruses (e.g.,influenza A, influenza B), respiratory syncytial viruses, Rotaviruses,Hantaviruses, human Papilloma viruses (e.g., HPV-16, HPV-18),Poxviruses, Poliovirus, rabies viruses, Retroviruses (e.g., HIV-1,HIV-2), hepatitis viruses (e.g., human HAV, HBV or HCV), Togaviruses andFlaviviruses (e.g., West Nile Virus, Yellow Fever Virus, Dengueviruses), Herpesviruses (e.g., CMV, EBV, Varicella Zoster Virus, HSV-1,HSV-2, HHV-6, HHV-8), measles viruses, mumps viruses, rubella virus,pneumococci such as Klebsiella pneumonia cells or capsule material,enteric bacterial cells (e.g., E. coli, or Shigella or Salmonellaspecies), gram positive bacterial pathogens (e.g., Staphylococcus,Streptococcus), gram negative bacterial pathogens, diphtheria pathogens,or human or animal cells obtained from a melanoma or skin cancer, breastcancer, prostate cancer, colon cancer, liver cancer, bone cancer,nervous tissue cancer (e.g., neuroblastoma, glioma), lymphoma, leukemiacells, kidney or renal cell cancer, ovarian cancer or lung cancer (e.g.,small cell carcinoma, non-small cell carcinoma). Exemplary parasites orantigen sources include Plasmodium, Leishmania and Cryptosporidium. Theantigen(s) that is used may comprise a pathogen coat protein(s),pathogen cell wall or other structural proteins. Antigen(s) from tumorcells or parasites may comprise cell membrane associated structures orintracellular molecules that are characteristic of, or unique to, thetumor or parasite. Other suitable antigen or pathogen sources are asdescribed herein or in the cited references.

Cancer and hyperproliferation conditions. Many cancers, precancers,malignancies or hyperproliferation conditions are associated with anunwanted Th2 immune response, a deficient Th1 response or unwantedinflammation. An insufficient Th1 immune response may play a role in thecapacity of malignant or premalignant cells to escape immunesurveillance. The formula 1 compounds, including those in the compoundgroups and embodiments disclosed herein, may thus be used to treat,prevent or slow the progression of one or more cancers, precancers orcell hyperproliferation conditions or they may be used to ameliorate oneor more symptoms thereof. In these conditions, the formula 1 compoundsare useful to enhance the subject's Th1 responses or to reestablish amore normal Th1-Th2 balance in the subject's immune responses. Theformula 1 compounds may function at least in part by decreasinginflammation or inflammation associated markers such as IL-6 and/or byenhancing hemopoiesis in many of these conditions.

These conditions include cancers or precancers comprising carcinomas,sarcomas, adenomas, blastoma, disseminated tumors and solid tumors suchas one associated with or arising from prostate, lung, breast, ovary,skin, stomach, intestine, pancreas, neck, larynx, esophagus, throat,tongue, lip, oral cavity, oral mucosa, salivary gland, testes, liver,parotid, biliary tract, colon, rectum, cervix, uterus, vagina, pelvis,endometrium, kidney, bladder, central nervous system, glial cell,astrocyte, squamous cell, blood, bone marrow, muscle or thyroid cells ortissue. The formula 1 compounds are thus useful to treat, prevent, slowthe progression of, or ameliorate one or more symptoms of a precancer,cancer or related hyperproliferation condition such as myelodysplasticsyndrome, actinic keratoses, endometriosis, Barrett's esophagus,leiomyoma, fibromyoma, benign or precancerous intestinal or bowel polypsor benign prostatic hyperplasia. The compounds can also be used totreat, prevent, slow the progression of, slow the replication or growthof, or to ameliorate one or more symptoms of a primary tumor, ametastasis, an advanced malignancy, a blood born malignancy, a leukemiaor a lymphoma.

The formula 1 compounds can be used to treat paraneoplastic syndromes orconditions such as ones associated with lung or breast cancers thatsecrete calcitonin or that enhance osteoclast activity. Such conditionsinclude hypercalcemia, Cushing's syndrome, acromegaly and non-islet celltumor hypoglycemia. The compounds are used to decrease osteoclastactivity or other symptoms associated with such conditions.

Hyperproliferation conditions that can be treated include melanoma,Kaposi's sarcoma, leiomyosarcoma, non-small cell lung cancer, small celllung cancer, bronchogenic carcinoma, renal cell cancer or carcinoma,glioma, glioblastoma, pancreatic or gastric adenocarcinoma,gastrointestinal adenocarcinoma, human papillomavirus associatedcervical intraepithelial neoplasia, cervical carcinoma, hepatoma,hepatocellular carcinoma, hepatocellular adenoma, cutaneous T-celllymphoma (mycosis fungoides, Sezary syndrome), colorectal cancer,chronic lymphocytic leukemia, chronic myelogenous leukemia, ALL orfollicular lymphoma, multiple myeloma, carcinomas with p53 mutations,colon cancer, cardiac tumors, adrenal tumors, pancreatic cancer,retinoblastoma, a small cell lung cancer, a non-small cell lung cancer,intestinal cancer, testicular cancer, stomach cancer, neuroblastoma,neuroma, myxoma, myoma, endothelioma, osteoblastoma, osteoclastoma,osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer,Kaposi's sarcoma, ovarian cancer, squamous cell carcinoma of thegastrointestinal tract. Treating a subject with a formula 1 compound canameliorate one or more side effects of chemotherapy or cancer symptomssuch as alopecia, pain, fever, malaise, chronic fatigue and cachexia orweight loss. Other cancers, precancers or their symptoms that can betreated, prevented or ameliorated are described in, e.g., Holland•FreiCancer Medicine ^(e.)5, 5^(th) edition, R. C. Bast et al., editors,2000, ISBN 1-55009-113-1, pages 168-2453, B. C. Becker Inc. Hamilton,Ontario, Canada or The Merck Manual, 17^(th) edition, M. H. Beers and R.Berkow editors, 1999, Merck Research Laboratories, Whitehouse Station,N.J., ISBN 0911910-10-7.

In some of these embodiments, the subject's hyperproliferation ormalignant condition may be associated with or caused by one or morepathogens. Such conditions include hepatocellular carcinoma associatedwith HCV or HBV, Kaposi's sarcoma associated with HIV-1 or HIV-2, T cellleukemia associated with HTLV I, Burkitt's lymphoma associated withEpstein-Barr virus or papillomas or carcinoma associated with papillomaviruses (e.g., human HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 45) orgastric adenocarcinoma, gastric MALT lymphoma or gastric inflammationassociated with Helicobacter pylori, lactobacillus, enterobacter,staphylococcus or propionibacteria infection.

In some of these embodiments, the formula 1 compounds may be used totreat, prevent or slow the progression of or ameliorate one or moreconditions in a subject having or subject to developing ahyperproliferation condition where angiogenesis contributes to thepathology. Abnormal or unwanted angiogenesis or neovascularizationcontributes to the development or progression of solid tumor growth andmetastases, as well as to arthritis, some types of eye diseases such asdiabetic retinopathy, retinopathy of prematurity, macular degeneration,corneal graft rejection, neovascular glaucoma, rubeosis, retinoblastoma,uvietis and pterygia or abnormal blood vessel growth of the eye, andpsoriasis. See, e.g., Moses et al., Biotech. 9:630-634 1991, Folkman etal., N. Engl. J. Med., 333:1757-1763 1995, and Auerbach et al., J.Microvasc. Res. 29:401-411 1985.

Dosages of the formula 1 compound, routes of administration and the useof combination therapies with other standard therapeutic agents ortreatments could be applied essentially as described above for cancer orhyperproliferation conditions or other conditions as disclosed herein.This, in some embodiments, the use of the formula 1 compound isoptionally combined with one or more additional therapies for a canceror precancer(s), e.g., one or more of surgery and treatment with anantiandrogen or an antiestrogen as described herein or in the citedreferences, an antineoplastic agent such as an alkylating agent, anitrogen mustard, a nitrosourea, an antimetabolite or cytotoxic agent,or an analgesic such as propoxyphene napsylate, acetaminophen orcodeine. Exemplary anticancer and adjunct agents include methotrexate,thioguanine, mercaptopurine, adriamycin, chlorambucil, cyclophosphamide,cisplatin, procarbazine, hydroxyurea, allopurinol, erythropoietin,G-CSF, bicalutamide, anastrozole, fludarabine phosphate and doxorubicin.Such therapies would be used essentially according to standard protocolsand they would precede, be essentially concurrent with and/or followtreatment with a formula 1 compound. In some embodiments, suchadditional therapies will be administered at the same time that aformula 1 compound is being used or within about 1 day to about 16 weeksbefore or after at least one round of treatment with the formula 1compound is completed. Other exemplary therapeutic agents and their usehave been described in detail, see, e.g., Physicians Desk Reference54^(th) edition, 2000, pages 303-3250, ISBN 1-56363-330-2, MedicalEconomics Co., Inc., Montvale, N.J. One or more of these exemplaryagents can be used in combination with a formula 1 compound toameliorate, slow the establishment or progression of, prevent or treatany of the appropriate cancers, precancers or related conditionsdescribed herein, or any of their symptoms.

In treating cancers or hyperproliferation conditions, the formula 1compounds may detectably modulate, e.g., decrease or increase, theexpression or level or activity of one or more biomolecules associatedwith the prevention, establishment, maintenance or progression of thecancer or hyperproliferation condition. Such biomolecules include one ormore of carcinoembryonic antigen, prostate specific antigen, her2/neu,Bcl-XL, bcl-2, p53, IL-1a, IL-1β, IL-6, or TNFα, GATA-3, COX-2, NFκB,IkB, an IkB kinase, e.g., IkB kinase-α, IkB kinase-β or IkB kinase-γ,NFAT, calcineurin, calmodulin, a ras protein such as H-ras or K-ras,cyclin D, cyclin E, xanthine oxidase, or their isoforms, homologs ormutant forms, which may have either reduced or enhanced biologicalactivity(ies), and which may be detectably decreased. Biomolecules ortheir activity(ies) that can be detectably increased include IL-2, IFNγ,IL-12, T-bet, O6-methylguanine-DNA-methyltransferase, calcineurin,calmodulin, a superoxide dismutase (e.g., Mn, Zn or Cu), a tumorsuppressor protein such as the retinoblastoma protein (Rb) or CDKN2A(p16), BRCA1, BRCA2, MeCP2, MBD2, PTEN, NBR1, NBR2 or the isoforms,homologs or mutant forms, which may have either attenuated or enhancedbiological activity(ies), of any of these molecules. One or more ofthese biomolecules may be modulated in any the cancers or conditionsdescribed herein.

The formula 1 compounds can modulate the synthesis or a biologicalactivity of one or more other gene products such as transcriptionfactors, enzymes or steroid or other receptors that are associated withthe establishment, progression or maintenance of a cancer or precanceror associated symptom. The compounds can inhibit AIB-1 coactivator orHER2/neu synthesis or activity in breast cancer cells or breast cancerconditions. They can enhance the synthesis or an activity of an estrogenreceptor such as ERα, ERβ1 or ERβ2 or progesterone receptor in breastcancer or colon cancer cells or conditions. These effects can includemodulation of the expression or one or more biological activities ofproteins or enzymes that contribute to disease establishment orprogression. Thus, the compounds can decrease IL-4, IL-6 or IL-13expression by stromal cells or immune cells that are in proximity to oradjacent to solid or diffuse tumor cells in a subject such as a human oranother mammal. In the cancers or precancers described herein, thecompounds can thus directly or indirectly modulate (e.g., decrease) theactivity or expression of relevant enzymes such as STAT-6, neutralendopeptidase, a hydroxysteroid dehydrogenase, such as a17β-hydroxysteroid dehydrogenase or a 3β-hydroxysteroid dehydrogenase.

In an exemplary embodiment, human patients suffering from melanoma ormelanoma precursor lesions are treated with a topical cream formulationcontaining 2-20% BrEA (w/w). The cream is applied to primary nevi(dysplastic nevi or common acquired nevi), primary cutaneous melanomas,secondary cutaneous melanomas and the skin surrounding the nevi ormelanomas. The areas to be treated are washed with soap or swabbed withan alcohol (e.g., ethanol or isopropanol) prior to administering thecream, when this is practical. About 0.1-0.4 g of cream, depending onthe size of the treated area, is applied once or twice per day pertreated region or lesion for about 10-20 days. The cream is leftundisturbed at the administration site for about 15-30 minutes beforethe patient resumes normal activity. Progression of the nevi andmelanomas is retarded in the majority of patients and significantregression is observed for some lesions. Following initial treatment,the formulation is administered every other day for at least 1 to 4months using the same dosing described for the initial round oftreatment. For these patients, standard therapy to treat precursorlesion or melanoma, e.g., dimethyl triazeno imidazole carboxamide ornitrosoureas (e.g., BCNU, CCNU), is optionally started or continuedaccording to the recommendations of the patient's doctor and with thepatient's informed approval. In cases where a tumor or precursor lesionis surgically removed and the site has sufficiently healed, the patientoptionally continues using the topical formulation at the site and theadjacent surrounding area every other day for at least 1 to 4 months. Insome of these embodiments, a formula 1 compound(s) is administered dailycontinuously as an oral composition or formulation, e.g., for a formula1 compound(s) that is a new compound per se. BrEA is optionally alsoadministered systemically using, e.g., a formulation described in theexamples below to deliver 0.1-5 mg/kg/day every other day for about 1week to about to 4 months, e.g., in the case of malignant melanoma.

Cardiovascular applications. The formula 1 compounds, including those inthe compound groups and embodiments disclosed herein, may be used totreat, prevent or slow the progression of one or more of congenitalheart defects, cardiovascular diseases, disorders, abnormalities and/orconditions, or to ameliorate one or more symptoms thereof in a subject.These include peripheral artery disease, arterio-arterial fistula,arteriovenous fistula, cerebral arteriovenous malformations, aorticcoarctation, cor triatum, coronary vessel anomalies, patent ductusarteriosus, Ebstein's anomaly, hypoplastic left heart syndrome,levocardia, transposition of great vessels, double outlet rightventricle, tricuspid atresia, persistent truncus arteriosus, and heartseptal defects, such as aortopulmonary septal defect, endocardialcushion defects, Lutembacher's Syndrome, ventricular heart septaldefects, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, post-infarction heartrupture, ventricular septal rupture, heart valve diseases, myocardialdiseases, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, cardiovascularsyphilis, cardiovascular tuberculosis, arrhythmias such as sinusarrhythmia, atrial fibrillation, atrial flutter, bradycardia,extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrialblock, long QT syndrome, parasystole, sick sinus syndrome, ventricularfibrillations, tachycardias such as paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia andheart valve diseases such as aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

The formula 1 compounds can be used to treat, prevent or ameliorate oneor more symptoms of myocardial diseases or pathological myocardial orvascular conditions such as alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, myocardial fibrosis,endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury,myocarditis, cardiovascular or vascular diseases such as dissectinganeurysms, false aneurysms, infected aneurysms, ruptured aneurysms,aortic aneurysms, cerebral aneurysms, coronary aneurysms, heartaneurysms, and iliac aneurysms, angiodysplasia, angiomatosis, bacillaryangiomatosis, Sturge-Weber Syndrome, angioneurotic edema, aorticdiseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterialocclusive diseases, arteritis, enarteritis, polyarteritis nodosa,cerebrovascular diseases, disorders, and/or conditions, diabeticangiopathies, diabetic retinopathy, thrombosis, erythromelalgia,hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension,idiopathic pulmonary fibrosis, peripheral vascular diseases, phlebitis,pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome,retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome,telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,venous insufficiency and arterial occlusive diseases such asarteriosclerosis, intermittent claudication, carotid stenosis,fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoyadisease retinal artery occlusion, thromboangiitis obliterans oratherosclerosis, any of which may be at an early stage or at a moreadvanced or late stage.

The formula 1 compounds can also be used to treat, prevent or ameliorateone or more symptoms of cerebrovascular diseases, thrombosis, and/orconditions such as carotid artery diseases, cerebral amyloid angiopathy,cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarteriovenous malformation, cerebral artery diseases, cerebral embolismand thrombosis, carotid artery thrombosis, sinus thrombosis,Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subduralhematoma, subarachnoid hemorrhage, cerebral infarction, cerebralischemia (including transient), subclavian steal syndrome,periventricular leukomalacia, vascular headache, cluster headache,migraine, vertebrobasilar insufficiency, air embolisms, embolisms suchas cholesterol embolisms, fat embolisms, pulmonary embolisms or amnioticfluid embolism, thromoboembolisms, thrombosis such as coronarythrombosis, hepatic vein thrombosis, retinal vein occlusion, carotidartery thrombosis, sinus thrombosis, Wallenberg's syndrome, andthrombophlebitis.

The formula 1 compounds can also be used to treat, prevent or ameliorateone or more symptoms of vascular ischemia or myocardial ischemias,vasculitis and coronary diseases, including angina pectoris, coronaryaneurysm, coronary arteriosclerosis, coronary thrombosis, coronaryvasospasm, myocardial infarction and myocardial stunning, cerebralischemia, ischemic colitis, compartment syndromes, anterior compartmentsyndrome, myocardial ischemia, reperfusion injuries, peripheral limbischemia, aortitis, arteritis, Behcet's Syndrome, mucocutaneous lymphnode syndrome, thromboangiitis obliterans, hypersensitivity vasculitis,Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener'sgranulomatosis.

Exemplary symptoms that the use of the formula 1 compounds canameliorate include one or more of pain such as arm, jaw or chest pain,edema or swelling, high blood pressure, shortness of breath or dyspnea,e.g., on exertion or while prone, fatigue or malaise and low cardiacinjection fraction. In treating a cardiovascular condition in a subjector in improving one or more symptoms thereof, the formula 1 compoundsmay accomplish one or more of increasing cardiac ejection volume orfraction, decreasing levels of IL-6, decreasing levels of C reactiveprotein, fibrinogen, cardiac creatinine kinase, increasing fatty acidmetabolism or utlization by cardiac tissue, increasing carnitylpalmitoyl fatty acid transferase or other cardiac metabolic enzymes,activating potassium dependent calcium channels, vasodilating orenhancing oxygen delivery to ischemic tissues or decreasing levels ofscarring or plaque formation that occurs, e.g., after vascular damage.Symptoms associated with a cardiovascular condition such as ischemiathat can be ameliorated also include acidosis, expression of one or moreimmediate early genes in, e.g., glial cells, vascular smooth musclecells or endothelial cells, neuronal membrane depolarization andincreased neuronal extracellular calcium and glutamate concentration.Other biological effects associated with treatment using a formula 1compound may also be monitored, e.g., and increase or decrease of a cellsurface antigen, a cytokine or an interleukin as disclosed herein.

Useful biological effects of the formula 1 compounds in cardiovascularindications such as myocardial ischemias also include preventing orreducing heart or vascular cell death and subsequent fibrosis. Theseeffects are associated with a decreased oxidative capacity of heartcells or myocytes, which is associated with a decreased capacity of thecells to metabolize fatty acids efficiently. The compounds enhance fattyacid metabolism and ameliorate the deleterious effects of a limitedoxidative capacity.

The formula 1 compounds also can limit inflammation or cell injury thatis associated with ischemia or oxygen reperfusion after ischemia.Ischemia, which is a detrimental decrease in oxygenated blood deliveryto affected cells or tissues, may arise from a cardiovascular conditionor event such as an infarction, or from thermal injury or burns.Ischemia may also arise from accidental or surgical trauma. Reperfusionafter cells have become hypoxic for a sufficient period of time can leadto tissue or cell injury that varies from slight to lethal. Thecompounds can reduce cell or tissue injury or death associated withischemia and reperfusion, by, e.g., reducing inflammation or the levelof a molecule associated with inflammation. Thus, levels of aproinflammatory cytokine or molecule such as leukotriene B4, plateletactivating factor or levels of extracellular P-selectin may result fromadministration of a formula 1 compound to a subject who may experiencereperfusion injury. Thus, the compounds can reduce injury or death of,e.g., neuron, cardiac, vascular endothelium, myocardial, pulmonary,hepatic or renal cells or tissues. Without wishing to be bound by anytheory, the compounds may act in part by reducing one or more ofneutrophil activation, platelet activation, platelet aggregation,endothelial cell activation and neutrophil adherence or adhesion toendothelial cells in these conditions.

The use of any formula 1 compound or species in any genus of formula 1compounds disclosed herein to treat, prevent or ameliorate any of thesecardiovascular conditions or symptoms will generally use one or more ofthe routes of administration, dosages and dosing protocols as disclosedherein. Thus, in exemplary embodiments, about 0.5 to about 100 mg/kg orabout 1 to about 25 mg/kg, of the formula 1 compound will beadministered per day by an oral, buccal, sublingual or parenteral route.Such administration can be, e.g., daily for about 5 to about 60 days inacute conditions or it can be intermittent for about 3 months to about 2years or more for chronic conditions. Alternatively, intermittent dosingcan be used essentially as described herein for acute cardiovascularconditions. In conditions such as ischemia, administration of theformula 1 compound should generally occur before or as soon after theischemic event as possible, e.g., within about 6 hours of an ischemicevent or about 12-24 hours before an anticipated ischemic event.

In related embodiments, the use of the formula 1 compound is optionallycombined with one or more additional therapies for cardiovasculardisorders, e.g., vascular surgery, cardiac surgery, angioplasty, ortreatment with andrenergic blockers, coronary vasodilators, calciumchannel blockers, nitrates, angiotensin converting enzyme inhibitors,anti-hypertensives, anti-inflammatory agents, diuretics, anti-arrhythmiaagents, thrombolytic agents, enzyme inhibitors such ashydroxymethylglutaryl CoA reductase inhibitors or xanthine oxidaseinhibitors. Exemplary hydroxymethylglutaryl CoA reductase inhibitorsinclude statins such as mevastatin, lovastatin, pravastatin, simvastatinor compounds described in U.S. Pat. No. 4,346,227, 4,448,979, 4,739,073,5,169,857, 5,006,530 or 5,401,746.

Other additional therapies include treatment with one or more ofdigoxin, nitroglycerin, doxazosin mesylate, nifedipine, enalaprilmaleate, indomethicin, tissue plasminogin activator, urokinase,acetylsalicylic acid, allopurinol or the like. Any of such additionaltherapies would be used essentially according to standard protocols andsuch therapies would precede, be concurrent with or follow treatmentwith a formula 1 compound. In some embodiments, such additionaltherapies will be administered at the same time that a formula 1compound is being used or within about 1 day to about 16 weeks before orafter at least one round of treatment with the formula 1 compound iscompleted. Other exemplary therapeutic agents and their use have beendescribed in detail, see, e.g., Physicians Desk Reference 54^(th)edition, 2000, pages 303-3251, ISBN 1-56363-330-2, Medical EconomicsCo., Inc., Montvale, N.J. One or more of these exemplary agents can beused in combination with a formula 1 compound to treat any of theappropriate cardiovascular disorders described herein.

Applications in autoimmunity, allergy, inflammation and relatedconditions. As mentioned above, the formula 1 compounds, including thosein the compound groups and embodiments disclosed herein, may be used totreat, prevent or slow the progression of one or more autoimmuneallergic or inflammatory diseases, disorders, or conditions, or toameliorate one or more symptoms thereof in a subject. These diseases andconditions include Addison's Disease, autoimmune hemolytic anemia,antiphospholipid syndrome, acute or chronic rheumatoid arthritis andother synovial disorders, an osteoarthritis including post-traumaticosteoarthritis and hypertrophic pulmonary osteoarthropathy, psoriaticarthritis, polyarthritis, epichondylitis, type I diabetes, type IIdiabetes, rheumatic carditis, bursitis, ankylosing spondylitis, multiplesclerosis, a dermatitis such as contact dermatitis, atopic dermatitis,exfoliative dermatitis or seborrheic dermatitis, mycosis fungoides,allergic encephalomyelitis, autoimmune glomerulonephritis, Goodpasture'sSyndrome, Graves'Disease, Hashimoto's Thyroiditis, multiple sclerosis,myasthenia gravis, neuritis, bullous pemphigoid, pemphigus,polyendocrinopathies, purpura, Reiter's Disease, autoimmune thyroiditis,systemic lupus erythematosus, scleroderma, fibromyalgia, chronic fatiguesyndrome, autoimmune pulmonary inflammation, Guillain-Barre Syndrome,type 1 or insulin dependent diabetes mellitus, autoimmune inflammatoryeye disease, hepatitis C virus associated autoimmunity, postinfectiousautoimmunity associated with, e.g., virus or bacterial infection such asa parvovirus such as human parvovirus B19 or with rubella virus,autoimmune skin and muscle conditions such as pemphigus vulgaris,pemphigus foliaceus, systemic dermatomyositis or polymyositis or anotherinflammatory myopathy, myocarditis, asthma such as allergic asthma,allergic encephalomyelitis, allergic rhinitis, a vasculitis conditionsuch as polyarteritis nodosa, giant cell arteritis or systemicnecrotizing vasculitis, chronic and an acute or chronic inflammationcondition such as chronic prostatitis, granulomatous prostatitis andmalacoplakia, ischemia-reperfusion injury, endotoxin exposure,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, cachexia, sarcoidosis, inflammatory boweldisease, regional enteritis, ulcerative colitis, Crohn's disease,inflammatory bowel disease or inflammation associated with an infection,e.g., septic shock, sepsis, or systemic inflammatory response syndrome.Any of these diseases or conditions or their symptoms may be acute,chronic, mild, moderate, severe, stable or progressing before, during orafter the time administration of the formula 1 compound to a subjectsuch as a human, is initiated. In general, a detectable improvement isobserved in the subject within a period of about 3 days to about 12months after initiation of a dosing protocol, e.g., the severity of thedisease or condition will detectably decrease, the rate of progressionwill detectably slow or the severity of a symptom(s) will detectablydecrease.

As used herein, acute inflammation conditions are characterized as aninflammation that typically has a fairly rapid onset, quickly becomesmoderate or severe and usually lasts for only a few days or for a fewweeks. Chronic inflammation conditions as used herein are characterizedas an inflammation that may begin with a relatively rapid onset or in aslow, or even unnoticed manner, tends to persist for at least severalweeks, e.g., about 3-6 weeks, months, or years and may have a vague orindefinite termination. Chronic inflammation may result when theinjuring agent (or products resulting from its presence) persists in thelesion, and the subject's tissues respond in a manner (or to a degree)that is not sufficient to overcome completely the continuing effects ofthe injuring agent. Other exemplary conditions are described in, e.g.,Textbook of Autoimmune Diseases, R. G. Lahita, editor, LippincottWilliams & Wikins, Philadelphia, Pa., 2000, ISBN 0-7817-1505-9, pages175-851 and Rheumatology, 2^(nd) edition, J. H. Klippel et al., editors,1998, ISBN 0-7234-2405-5, volume 1, sections 1-5 and volume 2, sections6-8, Mosby International, London, UK.

A formula 1 compound can be used to inhibit or ameliorate one or moreinappropriate immune responses or their symptoms in autoimmunity,inflammation, allergy or related conditions. The effects of the formula1 compounds include detectably ameliorating one or more of (1) theproliferation, differentiation or chemotaxis of T cells, (2) reducingunwanted cytotoxic T cell responses, (3) reducing unwanted autoantibodyor other antibody synthesis, e.g., an unwanted IgA, IgE, IgG or IgM, inallergy, asthma or another autoimmune or inflammation condition, (4)inhibiting the development, proliferation or unwanted activity ofautoreactive T or B cells, (5) altering the expression of one or morecytokines, interleukins or cell surface antigens, e.g., a cytokine,interleukin or cell surface antigen described herein (decreasing IL-8 inan autoimmune condition, decreasing the level of acute phase proteinssuch as C reactive protein or fibrinogen in inflammation conditions, (6)decreasing eosinophilia in allergy conditions, (7) detectably decreasingthe level or activity of one or more of ICAM-1, IL-1α, IL-1β, TNFα orIL-6 in, e.g., inflammation conditions or in autoimmune conditions suchas an arthritis or a myocarditis condition such as osteoarthritis,rheumatoid arthritis, toxic myocarditis, indurative myocarditis oridiopathic myocarditis, (8) decreasing the level or biological activityof one or more of anti-islet antibody, TNF, IFN-γ, IL-1, an arthritissymptom(s), nephritis, skin rash, photosensitivity, headache frequencyor pain, migraine frequency or pain, abdominal pain, nausea or anti-DNAantibodies in, e.g., insulin dependent diabetes mellitus or anautoimmune or inflammation condition such as systemic lupuserythematosus, rheumatoid arthritis or Crohn's disease, (9) reducinginduction of arachidonic acid metabolism or reducing eicosanoidmetabolites such as thromboxanes or prostaglandins in, e.g.,inflammation, asthma or allergy, (10) reducing IL-4, IL-8 or IL-10synthesis, levels or activity in, e.g., allergy or inflammation such asidiopathic pulmonary fibrosis or allergic asthma or (11) reducing orinterfering with neutrophil chemotaxis by, e.g., reducing thioredoxinrelease from affected cells in conditions such as cancer, infections,inflammation or autoimmunity.

Exemplary symptoms that the use of the formula 1 compounds canameliorate in these autoimmune, inflammatory and allergy conditionsinclude one or more of pain such as shoulder, hip, joint, abdominal orspine pain, joint stiffness or gelling, bursitis, tendonitis, edema orswelling, fatigue or malaise, headache, dyspnea, skin rash, fever, nightsweats, anorexia, weight loss, skin or intestine ulceration, muscleweakness, pericarditis, coronary occlusion, neuropathy and diarrhea. Intreating one of these conditions in a subject or in improving one ormore symptoms thereof, the formula 1 compounds may accomplish one ormore of decreasing levels of one or more of IL-1, IL-4, IL-6 or TNFα,decreasing levels of C reactive protein, fibrinogen or creatininekinase. Other biological effects associated with treatment using aformula 1 compound may also be monitored or observed, e.g., an increaseor decrease of a cell surface antigen, a cytokine or an interleukin asdisclosed herein.

In treating inflammation or any condition described herein whereinflammation contributes to the condition, the formula 1 compounds maydetectably modulate, e.g., decrease or increase, the expression or levelor activity of one or more biomolecules associated with the prevention,establishment, maintenance or progression of the inflammation condition.Such biomolecules include one or more of carcinoembryonic antigen,prostate specific antigen, her2/neu, Bcl-XL, bcl-2, p53, IL-1α, IL-1β,IL-6, or TNFα, GATA-3, COX-2, NFκB, IkB, an IkB kinase, e.g., IkBkinase-α, IkB kinase-β or IkB kinase-γ, NFAT, a ras protein such asH-ras or K-ras, cyclin D, cyclin E xanthine oxidase, or their isoforms,homologs or mutant forms, which may have either reduced or enhancedbiological activity(ies), and which may be detectably decreased.Biomolecules that can be detectably increased include IL-2, IFNγ, IL-12,T-bet, O6-methylguanine-DNA-methyltransferase, calcineurin, calmodulin,a superoxide dismutase (e.g., Mn, Zn or Cu), a tumor suppressor proteinsuch as the retinoblastoma protein (Rb) or CDKN2A (p16), BRCA1, BRCA2,MeCP2, MBD2, PTEN, NBR1, NBR2 or the isoforms, homologs or mutant forms,which may have either attenuated or enhanced biological activity(ies),of any of these molecules. One or more of these biomolecules may bemodulated in any inflammation condition described herein.

The use of any formula 1 compound or species in any genus of formula 1compounds disclosed herein to treat, prevent or ameliorate any of theseautoimmune, inflammatory or allergy conditions or symptoms willgenerally use one or more of the routes of administration, dosages anddosing protocols as disclosed herein. Thus, in exemplary embodiments,about 0.5 to about 100 mg/kg or about 1 mg/kg to about 15 mg/kg, of theformula 1 compound will be administered per day by, e.g., an oral,buccal, sublingual, topical or parenteral route. Such administration canbe, e.g., daily for about 5 to about 60 days in acute conditions or itcan be intermittent for about 3 months to about 2 years or more forchronic conditions. Alternatively, intermittent dosing can be usedessentially as described herein for acute autoimmune, inflammatory andallergy conditions.

In related embodiments, the use of the formula 1 compound is optionallycombined with one or more additional therapies for an autoimmune,inflammatory or allergy disorder(s), e.g., one or more of surgery andtreatment with a corticosteroid or glucocorticoid such ashydrocortisone, hydrocortisone acetate, prednisone, prednisolone,prednisolone acetate, methylprednisolone, dexamethasone, dexamethasoneacetate or triamcinolone acetonide, leflunomide, an antibody, e.g., ahuman or humanized monoclonal antibody, that decreases the activity orlevel of C5 complement, TNFα or TNFα receptor, an antirheumatic drugsuch as methorexate, D-penicillamine, sodium aurothiomalate,sulfasalazine or hydroxychloroquine, immunosuppressive agents such as6-thioguanylic acid, chlorambucil, cyclophosphamide or cyclosporin, anon-steroidal antiinflammatory agent such as celecoxib, ibuprofin,piroxicam or naproxin, an antihistamine such as loratidine orpromethazine hydrochloride or an analgesic such as propoxyphenenapsylate, acetaminophen or codeine. Such therapies would be usedessentially according to standard protocols and such they would precede,be concurrent with or follow treatment with a formula 1 compound. Insome embodiments, such additional therapies will be administered at thesame time that a formula 1 compound is being used or within about 1 dayto about 16 weeks before or after at least one round of treatment withthe formula 1 compound is completed. Other exemplary therapeutic agentsand their use have been described in detail, see, e.g., Physicians DeskReference 54^(th) edition, 2000, pages 303-3267, ISBN 1-56363-330-2,Medical Economics Co., Inc., Montvale, N.J. One or more of theseexemplary agents can be used in combination with a formula 1 compound toameliorate, prevent or treat any of the appropriate autoimmune,inflammatory or allergy conditions or disorders described herein or anyof their symptoms.

Regeneration and wound healing. The formula 1 compounds can be used tofacilitate cell differentiation or proliferation where regeneration oftissues is desired. The regeneration of tissues could be used to repair,replace, protect or limit the effects of tissue damaged by congenitaldefects, trauma (wounds, burns, incisions, or ulcers), age, disease(e.g. osteoporosis, osteoarthritis, periodontal disease, liver failure),surgery, including cosmetic plastic surgery, fibrosis, reperfusioninjury, or systemic cytokine damage. Tissues for which regeneration maybe enhanced include organs (e.g., pancreas, liver, intestine, kidney,skin, endothelium, oral mucosa, gut mucosa), muscle (e.g., smooth,skeletal or cardiac), vasculature (including vascular and lymphatics),nervous tissue, hematopoietic tissue, and skeletal tissue (e.g., bone,cartilage, tendon, and ligament). These effects may be accompanied bydecreased scarring or an increased rate of healing.

The formula 1 compounds are thus useful to enhance healing or tissuerepair in a subject having a bone fracture(s), e.g., a simple orcompound skull, spine, hip, arm or leg bone fracture. Similarly, nerveor brain tissue treatment using a formula 1 compound allows treating,slowing the progression of, ameliorating or preventing diseases such ascentral and peripheral nervous system diseases, neuropathies, ormechanical and traumatic diseases, disorders, and/or conditions (e.g.,spinal cord disorders, head trauma, cerebrovascular disease, and stoke).The compounds are useful to treat diseases associated with peripheralnerve injuries, peripheral neuropathy (e.g., resulting from chemotherapyor other medical therapies), localized neuropathies, and central nervoussystem diseases such as Alzheimer's disease, Parkinson's disease,Huntington's disease and amyotrophic lateral sclerosis. The subjectsundergoing treatment in these conditions may be elderly, e.g., a humanat least 55, 60, 65 or 70 years of age. Where the condition is acute,e.g., a bone fracture or a burn, the treatment may compriseadministration of a formula 1 compound to the subject on a daily orintermittent basis for about 3 days to about 12 months, e.g.,administration for about 2-12 weeks beginning after the subject sustainsan injury.

Dosages of the formula 1 compound, routes of administration and the useof combination therapies with other standard therapeutic agents ortreatments could be applied essentially as described herein, e.g., forcardiovascular conditions or other conditions as disclosed herein.

Neurological conditions. Nervous system diseases, disorders, orconditions, which can be ameliorated, treated or prevented with any ofthe formula 1 compounds disclosed herein include, but are not limitedto, nervous system trauma or injury, and diseases or conditions whichresult in either a disconnection of axons, a diminution of neuronfunction or degeneration of neurons, demyelination or pain.

Nervous system lesions which may be treated, prevented, or amelioratedin a subject include but are not limited to, the following lesions ofeither the central (including spinal cord, brain) or peripheral nervoussystems: (1) ischemic lesions, in which a lack of oxygen in a portion ofthe nervous system results in neuronal injury or death, includingcerebral infarction or ischemia, or spinal cord infarction or ischemia,(2) traumatic lesions, including lesions caused by physical injury orassociated with surgery, for example, lesions which sever a portion ofthe nervous system, or compression injuries, (3) malignant lesions, inwhich a portion of the nervous system is destroyed or injured bymalignant tissue which is either a nervous system associated malignancyor a malignancy derived from non-nervous system tissue, (4) infectiouslesions, in which a portion of the nervous system is destroyed orinjured as a result of infection, for example, by an abscess orassociated with infection by human immunodeficiency virus, herpeszoster, or herpes simplex virus or with Lyme disease, tuberculosis,syphilis, (5) degenerative lesions, in which a portion of the nervoussystem is destroyed or injured as a result of a degenerative processincluding but not limited to degeneration associated with Parkinson'sdisease, Alzheimer's disease, Huntington's chorea, or amyotrophiclateral sclerosis (ALS), (6) lesions associated with nutritionaldiseases, disorders, and/or conditions, in which a portion of thenervous system is destroyed or injured by a nutritional disorder ordisorder of metabolism including but not limited to, vitamin B 12deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration, (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis, (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins, (9)demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, progressive multifocal leukoencephalopathy, and centralpontine myelinolysis or a myelopathy, e.g., diabetic meylopathy or atransverse myelopathy, (10) neurological conditions such as insomnia,epilepsy, schizophrenia, depression, addiction to a drug, substance suchas tobacco, nicotine, caffeine, alcohol, a barbiturate, a tranquilizer,a narcotic such as hydromorphone HCl, propoxyphene napsylate, meperidineHCl, codeine, cocaine, morphine, heroin or methadone and (11) cognitivedysfunction conditions or diseases such as one or more of impairedlong-term or short-term memory, impaired concentration impairedattention or impaired learning, where the cognitive dysfunctioncondition or disease is optionally associated with chemotherapy,radiation therapy or exposure, aging, trauma, e.g., CNS trauma, orneurodegeneration.

The formula 1 compounds are useful to ameliorate, treat or prevent theonset, severity or length of other neurological diseases or conditionssuch as headache or a migraine condition or symptom such as classicmigraine, cluster headache, abdominal migraine, common migraine,hemiplegic migraine, ocular migraine, fulminating migraine, complicatedmigraine or a symptom of any of these such as head pain, vertigo,nausea, vomiting or potophobia.

In some embodiments, the formula 1 compound is used to protect neuralcells from the damaging effects of cerebral hypoxia, cerebral ischemiaor neural cell injury associated with cerebral infarction, heart attack,stroke or elevated levels of glucocorticoids such as cortisol. Thecompounds are also useful for treating or preventing a nervous systemdisorder may be selected, e.g., by assaying their biological activity inpromoting the survival or differentiation of neurons. For example, andnot by way of limitation, compositions of the invention which elicit anyof the following effects are useful: (1) increased survival time ofneurons in culture, (2) increased sprouting of neurons in culture or invivo, (3) increased production of a neuron-associated molecule inculture or in vivo, e.g., dopamine or choline acetyltransferase oracetylcholinesterase with respect to motor neurons or (4) decreasedsymptoms of neuron dysfunction in vivo. Such effects may be measured byany method known in the art. Increased survival of neurons may bemeasured using known methods, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 1990); increased sproutingof neurons may be detected by methods known in the art, such as themethods set forth in Pestronk et al. (Exp. Neurol. 70:65-82 1980) orBrown et al. (Ann. Rev. Neurosci. 4:17-42 1981). Increased production ofneuron-associated molecules may be measured by, e.g., bioassay,enzymatic assay, antibody binding or Northern blot assay, usingtechniques known in the art and depending on the molecule to bemeasured. Motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability. In otherembodiments, motor neuron diseases, disorders, and/or conditions thatmay be treated, prevented, and/or ameliorated include diseases,disorders, and/or conditions such as infarction, infection, exposure totoxin, trauma, surgical damage, degenerative disease or malignancy thatmay affect motor neurons as well as other components of the nervoussystem, as well as diseases, disorders, and/or conditions thatselectively affect neurons such as amyotrophic lateral sclerosis, andincluding, but not limited to, progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, infantile andjuvenile muscular atrophy, poliomyelitis and the post polio syndrome,and hereditary motorsensory neuropathy.

In some conditions such as mood changes, depression anxiety, memory lossor motor function impairment, the formula 1 compounds can modulate oneor more biological activities of a transcription factor or a steroidreceptor such as ERα in tissue such as the hypothalamus or amygdala orERβ in tissue such as the hippocampus, thalamus or entorhinal cortex.

For any of these diseases, conditions or their associated symptoms, thepresence of the disease, condition or symptom may be determined bysuitable objective or subjective means, e.g., assays to detect tissuedamage, levels of diagnostic markers, or an etiological agent, patientquestionaires or behavior performance tests, measurement of a diagnosticmarker(s), e.g., an enzyme, hormone, cytokine or drug substance in bloodor tissue, electroencephalography, imaging methods such as X-ray, MRIscan or CAT scan, observation and diagnosis of clinical features orsymptoms or biopsy of affected tissue or cells, e.g., aspiration biopsy,needle biopsy, incision biopsy or punch biopsy of tissue or cells.Neurological conditions, diseases and symptoms, which the formula 1compounds can be used to treat or ameliorate and methods to diagnose andcharacterize such conditions or diseases have been described. See, e.g.,Ph. Demaerel, A. L. Baert et al., eds. Recent Advances in DiagnosticNeuroradiology (Medical Radiology: Diagnostic Imaging) 2001 SpringerVerlag, ISBN: 3504657231, W. G. Bradley et al., Neurology in ClinicalPractice: Principles of Diagnosis and Management 1995, see, e.g., vol. 1Ch. 1-55 and vol 2. Ch. 1-66, Butterworth-Heinemann Medical, ISBN0750694777, H. J. M. Barnett et al., eds. Stroke: Pathophysiology,Diagnosis and Management 3^(rd) edition, 1998, see, e.g., pages 10-1450,Churchill Livingstone, ISBN 0443075514, P. J. Vinken et al., eds.Neurodystrophies and Neurolipidoses 2^(nd) ed. 1996, see, e.g., pages8-780, Elsevier Science, ISBN 0444812857, P. L. Peterson and J. W.Phillis eds. Novel Therapies for CNS Injuries: Rationales and Results1995, see, e.g., pages 8-380, CRC Press, ISBN 0849376521, D. Schiffer,Brain Tumors: Pathology and Its Biological Correlates 2^(nd) ed. 1997,see, e.g., pages 5-450, Springer Verlag, ISBN 3540616225 and E.Niedermeyer and F. Lopes Da Silva, eds. Electroencephalography: BasicPrinciples, Clinical Applications and Related Fields 4^(th) ed. 1999see, e.g., pages 13-1238, Lippincott, Williams & Wilkins, ISBN0683302841. The use of the formula 1 compounds in these conditions isoptionally combined with one or more of the therapeutic treatments thatare described in these references. The formula 1 compound may beadministered before, during or after another treatment is employed totreat or ameliorate a given neurological disease, condition or symptom.

Dosages of the formula 1 compound, routes of administration and the useof combination therapies with other standard therapeutic agents ortreatments could be applied essentially as described above forcardiovascular conditions or as disclosed elsewhere herein. Thus, theformula 1 compounds may be administered prophylactically ortherapeutically in chronic conditions or they may be administered at thetime of or relatively soon after an acute event such as an epilepticseizure, onset of a migraine or occurrence of trauma, accidental head orcentral nervous system injury or a cereberal stroke or infarction. Foracute events, the formula 1 compounds may thus be administeredconcurrently, e.g., within about 15 minutes or about 30 minutes of theonset or occurrence of the acute event, or at a later time, e.g., atabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22,24, 26, 28, 30, 36, 42, 48, 54, 60, 72, 84, 96, 108 or 120 hours afterthe onset or occurrence of the acute event. The formula 1 compounds maythus be administered at about 6-120 hours, or about 8-48 hours, about10-24 hours or about 12-16 hours after an acute event starts or occurs.

Skin treatments. The affect of the formula 1 compounds on immunefunction permits their use to improve the function of organs or organsystems that rely on the optimal functioning of one or more immuneresponses. Thus, the formula 1 compounds can be administered to asubject to prevent, treat, ameliorate, slow the progression of orenhance the healing of certain skin conditions such as skininflammation, lesions, atrophy or rash. As used here, skin includesexternal skin and internal skin or surfaces such as oral, intestinal andrectal mucosa. These conditions include lesions, rashes or inflammationassociated with, e.g., burns, infections and the thinning or generaldegradation of the dermis often characterized by a decrease in collagenor elastin as well as decreased number, size and doubling potential offibroblast cells. Such skin conditions include keratoses such as actinickeratosis, psoriasis, eczema, warts such as papillomavirus-inducedwarts, ulcers or lesions such as herpesvirus-induced ulcers or lesionsor diabetes associated ulcers or lesions, discoid lupus erythematosus,erythema nodosum, erythema multiform, cutaneous T cell lymphoma, atopicdermatitis, inflammatory vasculitis, relapsing polychondritis,exfoliative dermatitis, sarcoidosis, burns, melanoma, rash or irritationfrom poison oak, poison ivy or poison Sumac, blemished or hyperpigmentedskin, hyperkeratotic skin, dry skin, dandruff, acne, inflammatorydermatoses, scarring such as from a chemical or thermal burn andage-related skin changes. In these embodiments, treatment with theformula 1 compounds is optionally combined with other appropriatetreatments or therapies essentially as described herein, e.g., one ormore of a corticosteroid such as hydrocortisone or cortisol, prednisone,or prednisolone, an α-hydroxybenzoic acid or an α-hydroxycarboxylicacid(s) is coadministered with a formula 1 compound to treat, prevent orameliorate a skin condition such as atrophy or a lesion.α-Hydroxybenzoic acids and α-hydroxycarboxylic acids suitable for use inthese embodiments are described in, e.g., U.S. Pat. Nos. 5,262,407,5,254,343, 4,246,261, 4,234,599 and 3,984,566. The formula 1 compoundcan be used to minimize cutaneous atrophy caused by corticosteroids, aside-effect of their application to the skin.

In these embodiments that address skin conditions, dosages, routes ofadministration and dosing protocols for the formula 1 compounds areessentially as described herein. In some embodiments, the formula 1compound is administered to the subject in the form of a topical cream,ointment, spray, foam or gel. These topical formulations will optionallycomprise about 0.1% w/w to about 20% w/w, or about 0.2% w/w to about 10%w/w of a formula 1 compound in a composition that comprises one or moreexcipients that are suitable for such topical formulations, including,e.g., one or more agents that enhance penetration or delivery of theformula 1 compound into the skin. Such topical formulations can beadministered, e.g., once, twice or three times per day using about 0.1 gto about 8 g or about 0.2 g to about 5 g of the topical formulation oneach occasion. Administration may be daily for about 1 to about 28 days,or it may be intermittent and used as needed. The amount of a topicalformulation that can be administered may be higher, e.g., about 15 g orabout 20 g, if the size of the area to be treated is relatively large,e.g., at least about 30 cm² to about 100 cm² or more. Alternatively,systemic administration of the formula 1 compound such as oral,parenteral, sublingual or buccal delivery may be used, particularly whenthe area of the skin to be treated is relatively large. In some cases,both topical and systemic administration of a formula 1 compound can beused. Excipients that topical or other formulations may contain includethose described herein, or agents that enhance permeation orsolubilization of the formula 1 compound, e.g., DMSO or an alkylalkanol,such as a 2-alkylalkanol or a 3-alkyloctanol that comprises about 8-36carbon atoms (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20carbon atoms) such as 2-ethyloctanol, 2-propyloctanol, 2-octyldodecanol,2-butyloctanol, 2-hexyldecanol, 2-pentylnonanol, 3-ethyloctanol,3-propyloctanol, 3-octyldodecanol, 3-butyloctanol, 3-hexyldecanol,3-pentylnonanol, isostearyl alcohol, isocetyl alcohol, or mixturesthereof. Such alkylalkanol moieties include those having the structureHO—CH₂—(CH₂)₀₋₄—CH(C1-10 alkyl)-(CH₂)₀₋₆—CH₃, any of which areoptionally substituted at the alkanol or the alkyl moiety with one, two,three or more independently selected substituents as described herein,e.g., with one, two, three or more independently selected —O—, —F, —OH,—CN or —CH═CH— moieties. Such formulations can be used in therapeuticapplications described herein or in cosmetic applications.

Enhancement of hematopoiesis. The invention includes methods to treat orprevent various blood cell deficiencies such as TP or NP. Without beingbound to any theory, the treatment methods may at least in part resultin enhanced hematopoiesis (or hemopoiesis) or the treatment methods mayreduce the loss of cells such as platelets or neutrophils. Increasedplatelet or neutrophil production or reduced loss is typically observedas increased circulating blood cell counts. Thus, invention aspectscomprise methods to treat or prevent neutropenia in a subject in needthereof, comprising administering to a subject in need, or delivering tothe subject's tissues, an effective amount of a formula 1 compound.

Normal ranges of various white blood cells or blood components in adult(about 18-49 years of age) human blood are as follows. Total adult whiteblood cell counts average about 7500/mm³, with an approximate normalrange of about 4.5-11.0×10³/mm³. The normal basophil level is about 35mm⁻³, with a normal range of about 10-100/mm³. The normal adultneutrophil level is about 4400/mm³, with a normal range of about2000-7700/mm⁻³. The normal eosinophil level is about 275 mm⁻³, with anormal range of about 150-300/mm³. The normal monocyte level is about540 mm⁻³, with a normal range of about 300-600/mm³. The normal adultplatelet level is about 2.5×10⁵/mm³, with a normal range of about2.1×10⁵-2.9×10⁵/mm³. The normal adult red cell mass corresponds to about4.6×10¹² red cells/L in females and about 5.2×10¹² red cells/L in males.

Thus, a human patient in need of treatment will typically have, or besubject to, a cell count below these values. As used herein, neutropeniameans generally a circulating neutrophil count of less than about1800/mm³, generally a count of about 1500/mm³ or less. Thrmobocytopeniagenerally means a circulating platelet count of less than about1.6×10⁵/mm³, less than about 1.5×10⁵/mm³, less than about 1.3×10⁵/mm³ orless than about 1.0×10⁵/mm³. Anemia generally means a red cell masscorresponding to less than about 4.0×10¹² red cells/L in adult femalesand less than about 4.5×10¹² red cells/L in adult males (a hemoglobinlevel of less than about 12.0 g/dL in adult females and less than about13.5 g/dL in adult males).

In some cases, the diagnosis of a deficiency may cover a cell count thatfalls outside these ranges, due, e.g., to individual variations in asubject's age, sex, race, animal strain or normal blood cell status forthe individual. Such variations are identified by known means such as byidentification of a change from the subject's normal status or bymultiple cell measurements over time that reveal a deficiency. See,e.g., Hematology—Basic Principles and Practice, 2^(nd) edition, R.Hoffman, E. J. Benz Jr. et al., editors, Churchill Livingstone, NewYork, 1995. Subjects with an identified or identifiable deficiencyoutside these standard ranges are included in the definition of a bloodcell deficiency or a subject in need of treatment, as used herein.

Specific conditions that are amenable to prophylaxis or treatment by theinvention methods include the acquired blood cell deficiencies.Exemplary deficiencies or groups of deficiencies are neonatal alloimmuneTP, immune TP, immune thrombocytopenic purpura, thromboticthrombocytopenic purpura, post-transfusion purpura, radiation associatedTP, chemotherapy associated TP (e.g., NSAID treatments such as withindomethicin, ibuprofen, naproxen, phenylbutazone, piroxicam orzompirac, or β-lactam antibiotic treatments such as with ampicillin,carbenicillin, penicillin G, ticarcillin, or cephalosporin treatmentssuch as with cefazolin, cefoxitin or cephalothin, anticoagulanttreatments such as heparin, hirudin, lepirudin or aspirin, treatmentwith plasma expanders or psychotropic drugs), amegakaryocitic TP,chemotherapy associated TP, radiation associated TP, TP associated withsolid organ allograft or xenograft rejection or immune suppressiontherapy in solid organ or other tissue transplants (e.g., liver, lung,kidney, heart, bone marrow, hematopoietic stem cell or endothelial celltransplant, implant or transfusion), cardiopulmonary bypass surgery orchemotherapy associated TP (e.g., an anticancer, antiviral,antibacterial, antifungal or antiparasite therapy), cardiovasculardisease or therapy associated TP (e.g., congenital cyanotic heartdisease, valvular heart disease, pulmonary embolism, pulmonaryhypertension disorders or diltiazem, nifedipine, nitroglycerin ornitroprusside therapy), TP associated with chronic or acute renalfailure or treatment for these conditions (e.g., dialysis), TPassociated with infection such as a virus or bacterial infection,postinfectious NP, drug-induced NP, autoimmune NP, chronic idiopathicNP, basophilic leukopenia, eosinophilic leukopenia, monocyticleukopenia, neutrophilic leukopenia, cyclic NP, periodic NP,chemotherapy associated NP, radiation associated NP, chemotherapyassociated NP, radiation associated NP, NP associated with solid organallograft or xenograft rejection or immune suppression therapy in solidorgan or other tissue transplants (e.g., liver, lung, kidney, heart,bone marrow, hematopoietic stem cell or endothelial cell transplant,implant or transfusion), chemotherapy associated leukopenia, radiationassociated leukopenia, leukopenia associated with solid organ allograftor xenograft rejection or immune suppression therapy in solid organ orother tissue transplants (e.g., liver, lung, kidney, heart, bone marrow,hematopoietic stem cell or endothelial cell transplant, implant ortransfusion), immune hemolytic anemias, anemia associated with chronicor acute renal failure or treatment for these conditions (e.g.,dialysis), anemia associated with chemotherapy (e.g., isoniazid,prednisone) or anemia associated with radiation therapy.

Some of the blood cell deficiencies are associated with, or caused by,other therapeutic treatments, e.g., cancer chemotherapy, anti-pathogenchemotherapy, radiation therapy and chemotherapy for suppression ofautoimmunity or immune suppression therapy for organ or tissuetransplantation or implantation. The formula 1 compounds are thus usefulto facilitate or speed up immune system recovery in autologous bonemarrow transplant or stem cell transplant situations. In many cases itwould be medically sound to continue the treatment associated withcausing or exacerbating the blood cell deficiency. Thus, one wouldgenerally conduct the invention methods with subjects who are undergoinganother therapy at the same time or near the same time, e.g., within afew days to within about 1-6 months. Such subjects typically will havean identified blood cell deficiency such as a NP or a TP, e.g., asdisclosed herein. However, the formula 1 compounds are generallysuitable for preventing the onset of such deficiencies, and they canthus be used prophylactically in these indications. The inventionincludes all of these embodiments.

In some embodiments, the invention method is accomplished using aneffective amount of one or more growth factors or cytokines as a meansto further enhance the effect of the formula 1 compounds for theirintended uses or to modulate their effects. Suitable growth factors andcytokines are as described herein or in the cited references. Forexample, when one administers the formula 1 compound to enhancegeneration of platelets in humans or other subjects, or their precursorcells such as CFU-GEMM, BFU-Mk, CFU-Mk, immature megakaryocytes ormature postmitotic megakaryocytes, one can also administer one or moreof G-CSF, GM-CSF, SCF, Steel factor (“SF”), leukemia inhibitory factor(“LIF”), interkeukin-1α, (“IL-1α”), IL-3, IL-6, IL-11, TPO, EPO, theirisoforms, their derivatives (e.g., linked to a PEG or fusions such asPIXY321) or their homologs for other species. Similarly, administrationof the formula 1 compound to enhance the generation or function ofmyelomonocytic cells such as neutrophils, basophils or monocytes inhumans or other subjects, one can also administer one or more of G-CSF,GM-CSF, M-CSF, LIF, TPO, SF, interleukin-1 (“IL-1”), IL-2, IL-3, IL-4,interleukin-5 (“IL-5”), IL-6, IL-11, interleukin-12 (“IL-12”),interleukin-13 (“IL-13”), FLT3 ligand, their isoforms, homologs orderivatives (e.g., linked to a PEG or fusions such as PIXY321) or theirhomologs for other species. To enhance generation of red cells or theirprecursor cells such as CFU-GEMM, BFU-E or CFU-E in humans being treatedwith a formula 1 compound, one can co-administer one or more of G-CSF,GM-CSF, IL-1, IL-3, IL-6, TPO, EPO, transforming growth factor-[3],their isoforms, their derivatives (e.g., linked to a PEG or fusions suchas PIXY321) or their homologs for other species. See, e.g.,Hematology—Basic Principles and Practice, 3^(rd) edition, R. Hoffman, E.J. Benz Jr. et al., editors, Churchill Livingstone, New York, 2000 (see,e.g., Chapters 14-17 at pages 154-260). The co-administration of suchfactors in these methods is intended to enhance the efficacy of theformula 1 compound treatment, which is optionally measured by takingsuitable blood or tissue, e.g., bone marrow, samples at one or moretimes before and after the compounds have been administered. Suchco-administration will generally be compatible with a subject'scondition and other therapeutic treatments. Co-administration of suchfactors can precede, be simultaneous with, or follow the times ofadministration of the formula 1 compound(s) to the subject. Dosages ofsuch growth factors would generally be similar to those previouslydescribed, e.g., typically an initial course of treatment comprisesadministering about 1.0 to about 20 μg/kg/d for about 1-10 days, or asdescribed in, e.g., Hematology—Basic Principles and Practice, 3^(rd)edition, R. Hoffman, E. J. Benz Jr. et al., editors, ChurchillLivingstone, New York, 2000 (see, e.g., Chapter 51 at pages 939-979 andthe references cited therein).

In cases where a subject's blood cell deficiency is caused by, orassociated with another therapy, the invention contemplates that theother therapy will continue, if this is reasonable under thecircumstances. The timing of other therapies can precede, besimultaneous with, or follow the times of administration of the formula1 compound(s) to the subject. For example, chemotherapy for somemalignancies is accompanied by myelosuppression or a deficiency in oneor more blood cell types, e.g., TP or NP. Continued treatment would becalled for in some cases, and then the invention methods would beemployed to deliver to the subject an effective amount of a formula 1compound. Thus, alkylating agents, antimicrotubule agents,antimetabolites, topoisomerase I or II inhibitors, or platinum compoundssuch as one or more of mechlorethamine, vincristine, vinblastine,bleomycin, doxorubicin, epirubicin, tamoxifen, cyclophosphamide,etoposide, methotrexate, ifosfamide, melphalan, chlorambucil, busulfan,carmustine, lomustine, streptozocin, dacarbazine, vinorelbine,paclitaxel (taxol), docetaxel, cytosine arabinoside, hydroxyurea,fludarabine, 2′-chlorodeoxyadenosine, 2′-deoxycoformycin, 6-thioguanine,6-mercaptopurine, 5-azacytidine, gemcitabine, arabinofuranosylguanine,daunorubicin, mitoxantrone, amsacrine, topotecan, irinotecan, cisplatin,carboplatin, pilcamycin, procarbazine, aspariginase, aminoglutethimide,actinomycin D, azathioprine and gallium nitrate may be administered inconjunction with administration of any formula 1 compound(s) that isdisclosed herein. Treatments with other therapeutic agents such asheparin or nucleoside analogs such as 3-thiacytosine, azidothymidine ordideoxycytosine, or other antimicrobials such as cephalosporin, quinine,quinidine, gold salts (e.g., aurothioglucose), a fluoroquinolone (e.g.,ciprofloxacin), clarithromycin, fluconazole, fusidic acid, gentamycin,nalidixic acid, penicillins, pentamidine, rifampicin, sulfa antibiotics,suramin or vancomycin may result in a blood cell deficiency(s) and theycan thus be combined with administration of a formula 1 compound totreat the deficiency, or to ameliorate a symptom thereof. Similarly,anti-inflammatory drugs (e.g., salicylates, entanercept (a dimericfusion comprising a portion of the human TNF receptor linked to the Fcportion of human IgG1 containing the C_(H)2 and C_(H)3 domain and hingeregions of IgG1) or a COX-2 inhibitor such as celexicob(4-5[-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazole-1-yl]benzenesulfonamide)or rofecoxib (4-[4-methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone) or anIL-1 receptor antagonist such as anakinra), cardiac drugs (e.g.,digitoxin), β-blockers or antihypertensive drugs (e.g., oxprenolol orcaptopril), diuretics (e.g., spironolactone), benzodiazepines, (e.g.,diazepam) or antidepressants (e.g., amitriptyline, doxepin). Any ofthese methods also optionally include co-administration of one or moreof the growth factors described above, e.g., IL-3, G-CSF, GM-CSF or TPO.

In some embodiments, the formula 1 compounds that are used to enhancehematopoiesis or to treat associated conditions such as a TP or a NPdisease or condition as disclosed herein, are characterized by having alack of appreciable androgenicity. In these embodiments, the formula 1compounds are characterized by having about 30% or less, about 20% orless, about 10% or less or about 5% or less of the androgenicity of anandrogen such as testosterone, testosterone proprionate,dihydrotestosterone or dihydrotestosterone proprionate as measured in asuitable assay using suitable positive and/or negative controls.Suitable assays for androgenicity of various compounds have beendescribed, e.g., J. R. Brooks, et al., Prostate 1991, 18:215-227, M.Gerrity et al., Int. J. Androl. 1981 4:494-504, S. S. Rao et al., IndianJ. Exp. Biol. 1969 7:20-22, O. Sunami et al., J. Toxicol. Sci. 200025:403-415, G. H. Deckers et al., J. Steroid Biochem. Mol. Biol. 200074:83-92. The androgenicity of the formula 1 compounds are optionallydetermined as described or essentially as described in one or more ofthese assays or any other assay. Thus, one such embodiment comprises amethod to enhance hematopoiesis or to treat TP or NP comprisingadministering to a subject in need thereof an effective amount of aformula 1 compound, or delivering to the subject's tissues an effectiveamount of a formula 1 compound, wherein the formula 1 compound has about30% or less, about 20% or less, about 10% or less or about 5% or less ofthe androgenicity of an androgen such as testosterone, testosteroneproprionate, dihydrotestosterone or dihydrotestosterone proprionate asmeasured in a suitable assay, e.g., as described in the citations above.In conducting such methods, the subjects, e.g., rodents, humans orprimates, are optionally monitored for e.g., amelioration, prevention ora reduced severity of a disease, condition or symptom. Such monitoringcan optionally include measuring one or more of cytokines (e.g., TNFα,IL-1β), WBCs, platelets, granulocytes, neutrophils, RBCs, NK cells,macrophages or other immune cell types, e.g., as described herein or inthe cited references, in circulation at suitable times, e.g., atbaseline before treatment is started and at various times aftertreatment with a formula 1 compound such as at about 2-45 days aftertreatment with a formula 1 compound has ended.

In related embodiments, the activity or numbers of neutrophils ormonocytes is enhanced by co-administering the formula 1 compound with anneutrophil or monocyte stimulator, which is an non-protein agent ormolecule that can stimulate the activity or number of neutrophils ormonocytes in a subject. This aspect of the present invention encompassesany technique to enhance neutrophil or monocyte counts or activity.Means to accomplish this include administering an effective amount of aformula 1 compound and an effective amount of one or more of lithium,e.g., in the form of a salt such as lithium carbonate or chloride,deuterium oxide, levamisole (an antihelminthic agent), lactoferrin,thyroxine, tri-iodothyroxine, anthrax toxin, ascorbic acid,I-palmitoyl-lysophosphatidic acid, a calcium ionophore, e.g., A23187,cytochalasin B, sodium butyrate, piracetamine, micronized L-arginine,hydroxyurea and a bacterial lipopolysaccharide.

The neutrophil or monocyte stimulator can be administered at varioustime relative to administration of the formula 1 compound, includingabout 2-4 hours to about 1 or 2 weeks before administering the formula 1compound and including administration that is essentially simultaneouswith administering the formula 1 compound. Typically a neutrophil ormonocyte stimulator will be dosed according to known methods, includingdaily dosing of about 0.01 mg/kg/day to about 25 mg/kg/day. For example,about 1 g/day of ascorbic acid (e.g., about 0.5 to about 1.5 g/day) canbe administered to humans. When deuterium oxide is used as a neutrophilor monocyte stimulator, liquid aqueous formulations may comprise aformula 1 compound and deuterium oxide in place of some or all of thewater. Naturally occurring water contains approximately 1 part ofdeuterium oxide per 6500 parts water. Thus, the water present in aformulation may comprise, e.g., at least 1 part D₂O in 6000 parts H₂O,or at least 1 part in 100 parts, or about 50 parts or more per 100 partsof water. These aqueous formulations may comprise one or more additionalexcipients such as a cyclodextrin such as hydroxypropyl-β-cyclodextrin.Formulations comprising cyclodextrin and deuterium oxide, or comprisingcyclodextrin, deuterium oxide and water, may thus comprise deuteriumoxide in an amount greater than 1 part per 6500 parts water, such as 1part deuterium oxide per 1-100 parts water, e.g., 50 parts deuteriumoxide per 100 parts water. The amount of cyclodextrin can be in therange of from about 2 to about 85 grams per liter of water and/ordeuterium oxide, such as in the range of from about 5 to about 70 gramsper liter of water and/or deuterium oxide, one example of a suitableamount being in the range of about 45 grams per liter of water and/ordeuterium oxide.

In conducting any of these methods, one can monitor the subject'sclinical condition at any relevant time before, during or afteradministration of the formula 1 compounds, which treatments areoptionally combined with any of the other agents or treatments disclosedherein, such as cytokines, interleukins or an agent or molecule that canstimulate the activity or number of neutrophils or monocytes. Thesubject's blood can be drawn on one, two or more occasions in advance oftreatment to, e.g., obtain a baseline or initial level of white or redblood cells, to verify a presumptive diagnosis of a blood celldeficiency or to determine a blood parameter such as circulatingmyelomonocyte counts, circulating neutrophil counts, circulatingplatelet counts or the myeloperoxidase index. Then, during the course oftreatment or thereafter the subject's blood can be drawn on one, two ormore occasions to follow the subject's response.

Invention embodiments include methods that comprise administering to asubject in need thereof an effective amount of a formula 1 compound andan effective amount of at least one form of interferon, such asγ-Interferon or a growth factor or interleukin such as G-CSF or IL-6.Interferons can enhance the biological activity of the white cells thatarise from increased hemopoiesis. This can be particularly useful whenthe subject's circulating blood cell deficiency is associated with,e.g., an infection or a chemotherapy that suppresses hemopoiesis.Administration of an growth factor or an interleukin such as IL-6 canfacilitate hemopoiesis by stimulating quiescent stem cells or otherprogenitors that give rise to deficient cell types. Related embodimentsreplace growth factor or interferon administration partially orcompletely by increasing endogenous production in the subject usingconventional methods, e.g., administering double stranded RNA tostimulate γ-IFN.

For cases where γ-IFN is administered, the administration is usuallyrelatively constant, e.g., daily. This is because in patients in whomγ-IFN is not generated endogenously in significant amounts, there is atendency for levels of γ-IFN to drop relatively quickly, i.e., withinone day. In other words, in a patient in whom initially, γ-IFN levelsare close to zero, it should be administered in an amount which iseffective to bring γ-IFN levels to within normal levels, e.g., up to 10nanograms per milliliter, and a similar amount of γ-IFN should beadministered each day thereafter.

Suitable forms of γ-IFN and their biological properties and methods toobtain them have been described, see, e.g., U.S. Pat. Nos. 4,289,690,4,314,935, 4,382,027, 4,376,821; 4,376,822, 4,460,685, 4,604,284 and5,145,677, European patent publication nos. EP 063,482 EP 088 540, andEP 087 686, N. Fujii et al., J. Immunol., 1983 130:1683-86. A. Zlotnicket al., J. Immunol. 1983 131:794-80, M. deLey et al., Eur. J. Immunol.1980 10:877-83, F. Dianzani et al., Infection and Immunity, 198029:561-63, G. H. Reem et al., Infection and Immunity 1982 37:216-21(1982), R. Devos et al., Nucleic Acids Research 1982 10(8):2487-501, G.Simons et al., Gene 1984 28:55-64, P. W. Gray et al., Nature, 1982295:503-508, D. Novick et al., EMBO Journal, 1983 2:1527-30.

An aspect of the invention is method to enhance hemopoiesis in a subjectin need thereof comprising administering to the subject, or deliveringto the subject's tissues, an effective amount of a compound offormula 1. In some embodiments, the formula 1 compound is not5-androstene-3β-ol-17-one, 5-androstene-3β,17β-diol,5-androstene-3β,7β,17β-triol or a derivative of any of these threecompounds that can convert to these compounds by hydrolysis. Exemplaryformula 1 compounds in this method include compounds wherein (1) one ortwo R¹⁰ at the 1, 4, 6, 8, 9, 12 and 14 positions is not —H, wherein theone or two R¹⁰ at the 1, 4, 6, 8, 9, 12 and 14 positions areindependently selected from —F, —Cl, —Br, —I, —OH, ═O, —CH₃, —C₂H₅, anether optionally selected from —OCH₃ and —OC₂H₅, and an ester optionallyselected from —O—C(O)—CH₃ and —O—C(O)—C₂H₅, and/or (2) R¹, R², R³ and R⁴are independently selected from —H, —OH, ═O, an ester and an ether. Inthese embodiments, the subject may have thrombocytopenia or neutropeniaor the subject's circulating platelets, red cells, mature myelomonocyticcells, or their precursor cells, in circulation or in tissue may bedetectably increased. In some cases the subject has renal failure. Thesemethods may further comprise the steps of obtaining blood from thesubject before administration of the formula 1 compound and measuringthe subject's white or red cell counts and optionally, on one, two,three or more occasions, measuring the subject's circulating white cellor red cell counts after administration of the formula 1 compound, e.g.,within about 12 weeks after an initial administration of a formula 1compound or during or within about 12 weeks after a course of treatment.Such a treatment course may as described herein.

In any of the methods disclosed herein, a treatment may be interruptedbriefly or for extended periods of time. The reason for suchinterruption can be any of a wide variety, e.g., patient non-compliance,apparent improvement in a subject's condition or by design. Any suchinterruption would not take a regimen outside the scope of the presentinvention. For example, a patient might miss a day or several days ofadministration. Similarly, the regimen might call for administration ofone or more compounds for one or more day, and then non-administrationof the one or more compounds for one or more day, and then resumption ofthe administration of the one or more compounds. Furthermore, a regimenaccording to the present invention can be altered in view of a patient'scurrent condition, and can continue for any length of time, includingthe entire subject's lifetime.

For administration of γ-IFN, a volume of about 1 mL of a solid or liquidsublingual formulation that comprises about 100 micrograms of γ-IFN maybe used. An exemplary liquid formulation comprises a saline solutioncontaining 45 weight % hydroxypropylcyclodextrin. It would be expectedthat such a dosage would provide in the range of 30 to 40 micrograms ofγ-IFN to the patient's blood. Such sublingual formulations would be heldunder the patient's tongue for a period of time sufficient to allow someor all of the γ-IFN to be delivered to the patient while held under thepatient's tongue. Such administration has not been previously known inthe art, in which conventionally, it has been thought thatadministration of γ-IFN must be by injection, e.g., subcutaneousinjection. Subcutaneous injection of γ-IFN is associated with unwantedside effects, including fatigue, headache, night sweats, fever, localpain at the injection site, nausea, vomiting, diarrhea and others. Theabove-described sublingual γ-IFN formulations of the present inventionis an aspect of the present invention, which can be of use in accordancewith other aspects of the present invention as described herein. Ingeneral, however, a wide variety of routes of administration could beemployed for γ-IFN in accordance with the present invention, includingthose disclosed in U.S. Pat. No. 5,145,677.

Modulation of transcription factors, receptors and gene expression. Intreating any of the diseases, conditions or symptoms disclosed herein,the formula 1 compounds can modulate, i.e., detectably enhance orincrease or detectably inhibit or decrease, the expression or abiological activity(ies) of one or more transcription factors orreceptors. This can lead to detectable modulation of target geneactivity or expression as part of the treatment or amelioration of thedisease, condition or symptom. Such modulation can arise from changes inthe capacity of a transcription factor or receptor to bind to or form acomplex with other natural ligands such as a target DNA sequence(s),another transcription factor(s), a transcription cofactor, a receptorsuch as a steroid receptor or cell membrane receptor (e.g., a lipid,peptide, protein or glycoprotein receptor such as an interleukinreceptor or a growth factor receptor), a receptor cofactor or an enzymesuch as a polymerase, kinase, phosphatase or transferase. The effects offormula 1 compounds on these biomolecules can be exerted in immune cellsor in non-immune tissue, e.g., cells or tissue adjacent to diseasedtissue such as infected or malignant cells. The formula 1 compounds maydirectly or indirectly modulate the capacity of any of these moleculesto transduce signals that are part of normal signal trandsuctionprocesses.

In many of the clinical conditions described herein, e.g., in cancers,infections, acute inflammation, chronic inflammation or autoimmunity,the formula 1 compounds can modulate, e.g., detectably decrease orincrease, a biological activity(ies), protein or molecule level or RNAlevel of 1, 2, 3, 4, 5, 6 or more biomolecules that are involved inestablishment, maintenance or progression of a disease, condition orsymptom. Such biomolecules include 1, 2, 3, 4, 5, 6 or more of AP-1, acyclooxygenase such as cyclooxygenase-1 (COX-1) or cyclooxygenase-2(COX-2), TNFα, TNFα receptor 1, TNFα receptor 2, TNF receptor-associatedfactor, TNFβ, TNFβ receptor, MIP-1α, monocyte chemoattractant-1 (MCP-1),interferon gamma (IFNγ or γIFN), IL-1α, IL-1β, IL-1α receptor, IL-1βreceptor, IL-2, IL-3, IL-4, IL-4 receptor (IL-4R), IL-5, IL-6, IL-6receptor (IL-6R), IL-8, IL-8 receptor (IL-8R), IL-10, IL-10 receptor(IL-10R), IL-12, an IL-12 receptor, (e.g., IL-12Rβ2), IL-13, IL-15,IL-17, IL-18, nuclear factor kappa B (NFκB), AP-1, c-maf, v-maf, mafB,NrI, mafK, mafG, the maf family protein p18, reactive oxygen species,e.g., hydrogen peroxide or superoxide ion (collectively ROS), a17β-hydroxysteroid dehydrogenase (17β-HSD) or an 11β-hydroxysteroiddehydrogenase (11β-HSD), e.g., 11β-HSD type 1, 11β-HSD type 2, 17β-HSDtype 1, 17β-HSD type 2 or 17β-HSD type 5, a steroid aromatase, e.g.,cytochrome P450 aromatase, steroid 5α-reductase, serum or blood cortisolcytosolic phospholipase A2 (cPLA2), calcium-independent phospholipase A2(iPLA2), a prostaglandin, e.g., prostaglandin E2 (PGE2) or prostaglandinD2 (PGD2), a leukotriene, e.g., leukotriene B4, inducible nitric oxidesynthetase (iNOS), nitric oxide (NO), GM-CSF, RANTES (regulated onactivation, normal T cells expressed and secreted), eotaxin, GATA-3,CCR1, CCR3, CCR4, CCR5, CXCR4, in, e.g., a subject's cell(s) ortissue(s) or in enzyme, tissue or cell-based assays. In these subjects,the levels of other biomolecules, their RNAs or the level of theiractivity can be detectably modulated include IFNα, INFα receptor, PPARα,PPARγ, PPARδ or a transcription factor such as T-bet is detectablyincreased. Other biomolecules or their polymorphs or homologs that theformula 1 compounds directly or indirectly modulate include one or moreof, e.g., Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Janus kinase 3(JAK3), signal transducer and activator of transcription 1 (STAT1),signal transducer and activator of transcription 2 (STAT2) and signaltransducer and activator of transcription 3 (STAT3). The formula 1compounds can modulate the other biologically active analogs of anythese enzymes, chemokines, cytokines, their receptors or ligands,including their polymorphs or homologs. In some cells or tissues, one ormore of these biomolecules may be detectably increased, while in othercells or tissues, the same biomolecule may be detectably decreased.Thus, the biomolecules that the formula 1 compounds can modulate, e.g.,detectably increase or decrease, include the intracellular orextracellular level or biological activity of one or more enzyme,cytokine, cytokine receptor, chemokine and/or chemokine receptor.

Additional exemplary mammalian or human and other biomolecules, e.g.,transcription factors or receptors, including orphan nuclear receptors,their homologs, isoforms and co-factors (e.g., co-repressors,co-activators, transcription factors, gene promoter regions orsequences) and related molecules that the formula 1 compounds candirectly or indirectly from complexes with, or modulate (detectablyincrease or decrease) the synthesis, level or one or more biologicalactivities of, include steroidogenic factor-1 (SF-1), steroidogenicacute regulatory protein (StAR), chicken ovalbumin upstreampromoter-transcription factor (COUP-TFI) and its mammalian homologs,silencing mediator for retinoid and thyroid hormone receptor (SMRT) andits mammalian homologs, sterol regulatory element binding protein(SREBP) 1a (SREBP-1a), SREBP-1c, SREPB-2, NF-E3, FKHR-L1, COUP-TFII andits mammalian homologs, IκB, IκBα, AML-3, PEBP2αA1, Osf2, Cbfa1, RUNX2,activating transcription factor 2 (ATF2), c-Jun, c-Fos, a mitogenactivated kinase (MAP) such as p38 or JNK or an isoform thereof, amitogen activated kinase kinase (MKK) or an isoform thereof, steroidreceptor coactivator-1 family (SRC-1, SRC-1/serum response factor),SRC-2, SRC-3, SET, nerve growth factor inducible protein B, StF-IT,NFAT, NFAT interacting protein 45 (NIP45), IkB, an IkB kinase, NFATp,NFAT4, an AP-1 family protein, p300, CREB, CREB-binding protein (CPB),p300/CBP, p300/CPB-associated factor, SWI/SNF and their human and otherhomologs, BRG-1, OCT-1/OAF, AP-1, AF-2, Ets, androgen receptorassociated protein 54 (ARA54), androgen receptor associated protein 55(ARA55), androgen receptor associated protein 70 (ARA70), androgenreceptor-interacting protein 3 (ARIP3), ARIP3/PIASx α complex, PIASx α,Miz1, Miz1/PIASx β complex, PIASx β, PIAS1, PIAS3, GBP, GBP/PIAS1complex, RAC3/ACTR complex, SRC-1α, receptor interacting protein-140(RIP-140), transcription factor activator protein-1, activationfunction-2, glucocorticoid receptor-interacting protein-1 (GRIP-1),receptor interacting protein-160 (RIP-160), suppressor of gal4D lesions(SUG-1), transcription intermediary factor-1 (TIF-1), transcriptionintermediary factor-2 (TIF-2), SMRT, N—CoR, N—CoA-1, p/CIP, p65 (ReIA),the 120 KD rel-related transcription factor, heat shock proteins (HSP)such as HSP90, HSP70 and HSP72, heat shock factor-1, Vpr encoded by thehuman immunodeficiency virus and its isoforms and homologs thereof,testicular orphan receptor TR2, thyroid hormone α1 (TR α1), retinoid Xreceptor α, TR α1/RXR α heterodimer, direct repeat-4 thyroid hormoneresponse element (DR4-TRE), an estrogen receptor (ER) such as ERα orERβ, estrogen receptor related receptor α (ERRα), estrogen receptorrelated receptor β (ERRβ), estrogen receptor related receptor γ (ERRγ),steroid xenobiotic receptor (SXR), hepatocyte nuclear factor 4 (HNF-4),hepatocyte nuclear factor 3 (HNF-3), liver X receptors (LXRs), LXRα,LXRβ, estrogen receptor α (ERα), constitutive androstane receptor-β(CAR-β), RXR/CAR-β heterodimer, short heterodimer partner (SHP; NROB2),SHP/ERα heterodimer, estrogen receptor β, SHP/ERβ heterodimer,testicular orphan receptor TR4, TR2/TR4 heterodimer, pregnane X receptor(PXR) and isoforms, cytochrome P-450 monooxygenase 3A4, including itsgene promoter region and isoforms thereof, HNF-4/cytochrome P-450monooxygenase 3A4 gene promoter region and isoforms complex, HIV-1 longterminal repeat (LTR), HIV-2 LTR, TR2/HIV-1 LTR complex, TR4/HIV-1 LTRcomplex, TR4/HIV-1 LTR complex, TR α1/TR4/HIV-1 LTR complex, TR2isoforms (TR2-5, TR7, TR9, TR11), DAX-1, DAX-1/steroidogenic acuteregulatory protein gene promoter region, RevErb, Rev-erbA α, Rev-erb β,steroid receptor coactivator amplified in breast cancer (AIB 1),p300/CREB binding protein-interacting protein (p/CIP), thyroid hormonereceptor (TR, T3R), thyroid hormone response elements (T3REs),retinoblastoma protien (Rb), tumor suppressor factor p53, transcriptionfactor E2F, mammalian acute phase response factor (APRF), constitutiveandrostane receptor (CAR), Xenopus xSRC-3 and mammalian (human)homologs, TAK1, TAK1/peroxisome proliferator-activated receptor α(PPARα) complex, PPARα/RXRα complex, peroxisome proliferator-activatedreceptor β (PPARβ), peroxisome proliferator-activated receptor γ(PPARγ), peroxisome proliferator-activated receptor δ (PPARδ), farnesoidX receptor, retina X receptor, TAK-1/RIP-140 complex, retinoic acidreceptor (RAR), RARβ, TR4/RXRE complex, SF-1/steroid hydroxylase genepromoter region, SF-1/oxytocin, including its gene promoter region, bileacid receptor (FXR), nuclear receptor corepressor (NcoR), liver receptorhomologous protein-1 (LRH-1; NR5A2), SF-1/ACTH receptor gene promoterregion, rat Ear-2 and mammalian homologs, human TR3 orphan receptor(TR3), RLD-1, OR-1, androgen receptor, glucocorticoid receptor, estrogenreceptor, progesterone receptor, mineralcorticoid receptor, aldosteronereceptor, E6-associated protein (E6-AP), OR1, OR1/RXRα complex, TIF-1,CBP/P300 complex, TRIP1/SUG-1 complex, RIP-140, steroid receptorcoactivator 1 (SRC1), SRC1α/P160 complex and TIF-2/GRIP-1 complex,RAR/N—CoR/RIP13 complex, RAR/SMRT/TRAC-2 complex and protein X ofhepatitis B virus. The homologs, orthologs and isoforms of thesetranscription factors, receptors and other molecules are included amongthe molecules that the formula 1 compounds can modulate the synthesis orone or more biological activities of. Such factors are biologicallyactive or function in one or more of a number of cell types such as Tcells, B cells, macrophages, dendritic cells, platelets, monocytes,neutrophils, neurons, epithelial cells, endothelial cells, cartilagecells, osteoblasts, osteoclasts, splenocytes, thymocytes and GALTassociated cells. Methods to identify these molecules and theirbiological activities have been described, e.g., U.S. Pat. Nos.6,248,781, 6,242,253, 6,180,681, 6,174,676, 6,090,561, 6,090,542,6,074,850, 6,063,583, 6,051,373, 6,024,940, 5,989,810, 5,958,671,5,925,657, 5,958,671, 5,844,082, 5,837,840, 5,770,581, 5,756,673, andPCT publication Nos. WO 00/24245, WO 0073453 and WO 97/39721.

In one aspect, the compounds are used to treat, prevent or to ameliorateconditions or symptoms that are associated with unwanted or expressionor activity of one or more of these molecules in conditions such as,e.g., acute inflammation, chronic inflammation or their symptoms, acuteallergy, chronic allergy or their symptoms, e.g., allergic rhinitis oracute or chronic asthma, psoriatic arthritis, osteoporosis,osteoarthritis, rheumatoid arthritis, neurological dysfunction or theirsymptoms, e.g., dementias such as Alzheimer's Disease, Parkinson'sDisease, or memory loss conditions, in osteoporosis or in cancer such asbreast cancer. The compounds can prevent NFκB from translocating fromthe cytoplasm into the nucleus and thus can increase the ratio ofcytoplasmic NFκB to nuclear NFκB. The formula 1 compounds may inhibitactivation of NFκB-mediated transcription while NFκB is bound to targetDNA sequences in the nucleus. Alternatively, the formula 1 compounds canactivate or enhance the expression of or one or more activity of atranscription factor such as T-bet in, e.g., a subject's cell(s) ortissue(s) or in enzyme or cell-based assays. In this aspect thecompounds are used to treat, prevent or to ameliorate conditions orsymptoms that are associated with deficient expression or activity ofT-bet in conditions such as immune dysfunction in an immunosuppressioncondition, aging, an infection, a cancer or precancer as describedherein or in the cited references.

Thus, in some embodiments, the level or a biological activity of 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of COX-2, IL-1β, TNFα, TNFαreceptor 1, TNFα receptor 2, TNF receptor-associated factor, MIP-1α,MCP-1, IFNγ, IL-4, IL-4R, IL-6, IL-6R, IL-8, IL-8R, IL-10, IL-10R, NFκB,IkBα, AP-1, GATA-3, 11β-HSD1, cPLA2, iPLA2, cortisol, ROS, PGE2, PGD2,leukotriene A4, leukotriene B4, leukotriene C4, iNOS or GM-CSF areoptionally measured and they are generally detectably reduced, e.g., RNAor protein levels are reduced by about 10-95% or about 20-95% or morecompared to suitable untreated controls. In these embodiments, the levelor a biological activity of 4, 5, 6 or more of IFNα, INFα receptor,IL-12, an IL-12 receptor, (e.g., IL-12Rβ2), PPARα, PPARγ, and T-bet areoptionally measured and they are generally detectably increased. In achronic infection condition, e.g., HIV in humans, autoimmunity, achronic fungal or parasite infection or in a precancer or cancercondition, e.g., benign prostatic hyperplasia, the progression of thecondition may be slowed over a period of 1, 2, 3, 4, 5 or more years. Inthese embodiments, the subject's condition becomes more manageable witha reduced incidence or severity of side effects, e.g., a detectablehalt, slowing, reversal or decreased incidence of wasting, dementia, CD4cell count decreases or viral load increases, which tend to occur overtime in HIV infected humans or a halt, slowing or reversal of pathogenor precancer or cancer cell replication. The detectable halt, slowing,reversal of the condition or decreased incidence of side effects can beobserved as a decrease of about 10% or more, e.g., about a 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or more decline.

These effects are typically observed after administration of aneffective amount of a formula 1 compound using, e.g., a method or doseessentially as disclosed herein. The simultaneous reduction of multiplebiomolecules provides a method to modulate immune responses bymodulating multiple pathways that lead to a common condition such asinflammation. This provides a method to treat or ameliorate, e.g., acuteor chronic inflammation, a cancer, an infection or a symptom associatedtherewith, or to slow the progression of or reduce the severity of theseconditions or their symptoms.

Previously described methods can be used to measure the amount, activityor cellular location of various biomolecules such as cytokines ortranscription factors. See, e.g., U.S. Pat. Nos. 6,107,034, 5,925,657,5,658,744, 4,016,043 and 3,850,752, S. Szabo et al., Cell 2000100:655-669, Y. Nakamura et al., J. Allergy Clin. Immunol. 1999 103(2pt. 1):215-222., R. V. Hoch et al., Int. J. Cancer 1999 84:122-128.These methods can be used to measure the effects of the formula 1compounds on transcription factors or receptors in cells or tissues thathave been exposed to the compounds.

Without wishing to be bound to any theory, the formula 1 compounds maymodulate multiple biomolecules in a microenvironment sensitive manner orcontext. The effects of the compounds can provide a decrease in aparticular molecule such as IFNγ and a decrease in inflammationassociated with elevated IFNγ levels or activity without eliminatingbeneficial effects of the molecule. This effect arises from decreasingthe level or activity of a biomolecule such as IFNγ in cells that aredysregulated, while allowing normal immune cells to produce sufficientamounts of the same molecule to perform normal immune functions. Inlocations where the biomolecule is needed for activity, e.g., in lymphnodes or spleen cells, sufficient amounts of the modulated molecule arepresent to elicit a desired response, while the level of the molecule incells in circulation decreases. The compounds can increase IL-13, IL-15,IL-17 or IL-18 in conditions where a subject has a deficient Th1 immuneresponse, e.g., in infection or cancer. Conversely, the compounds candecrease IL-13, IL-15 or IL-18 in conditions such as allergy orautoimmune conditions, e.g., multiple sclerosis, where an excess Th1immune status may prevail.

In general, the formula 1 compounds will detectably decrease thesynthesis or one or more biological activity of one or more of thesemolecules (or other transcription factors or receptors disclosed herein)when such synthesis or activities is associated with the establishment,maintenance, progression or enhanced severity of a clinical condition orsymptom disclosed herein. Conversely, the formula 1 compounds willgenerally detectably increase the synthesis or one or more biologicalactivities of one or more of these molecules (or other transcriptionfactors or receptors disclosed herein) when such synthesis or activityis associated with the treatment, prevention, cure or amelioration of aclinical condition or symptom disclosed herein.

These decreases or increases compared to suitable controls can berelatively small, including changes near the lower limits ofdelectability for such molecules using known or new assays, e.g., adecrease or increase in the synthesis or biological activity of at leastabout 2%, about 5%, about 10% or about 20%. Such changes can be modestor relatively large, e.g., at least about a 50% change, at least about a90% change, or at least about a 200% change, up to about a 5-fold, abouta 10-fold, about a 100-fold or greater decrease or increase in thesynthesis or biological activity of the affected molecule(s) compared tosuitable controls. These changes are typically measured relative tocontrols that lack a formula 1 compound or that use known agonists orantagonists of one or more relevant molecules. Assays can be based onmeasuring decreases or increases in, e.g., one or more of proteinlevels, RNA or mRNA levels, a ligand binding activity, transcription ofa target gene(s) and the like. Suitable assay protocols include anysuitable polymerase chain reaction assay to measure an RNA or mRNA, anysuitable blotting protocol for nucleic acid or for protein such as aNorthern or Western blot method or any transcription assay, includingDNA footprinting or a gene expression or gene function assay. Typicallythe formula 1 compounds will effect detectable changes in the synthesisor one or more biological activities in a concentration range of about0.5×10⁻⁹ M to about 3×10⁻⁵ M. Exemplary compositions that comprise aformula 1 compound for use in, e.g., in vivo animal assays, in vitrocell or tissue culture assays or in cell free assays, will comprise oneor more suitable solvents or vehicles including DMSO, ethanol, water andtissue culture medium, which optionally contains calf, horse or goatserum or another serum.

One or more of these transcription factors, receptors or complexes canbe a component in methods when, e.g., they are used with a formula 1compound in cell-free assays or in tissue culture assays. Formation ofthese complexes in cells or analysis of the effects of formula 1compounds on one or more of their biological activities is facilitatedby inserting into the cells a DNA construct(s) that expresses one ormore of these proteins, e.g., mammalian or yeast cells containing astable DNA construct or a construct used for transient transfectionassays. Methods to perform assays or to induce biological responses invitro or in vivo using the formula 1 compounds as agonists, antagonistsor as reference standards are essentially as described, see, e.g., U.S.Pat. Nos. 5,080,139, 5,696,133, 5,932,431, 5,932,555, 5,935,968,5,945,279, 5,945,404, 5,945,410, 5,945,412, 5,945,448, 5,952,319,5,952,371, 5,955,632, 5,958,710, 5,958,892 and 5,962,443; InternationalPublication Numbers WO 96/19458, WO 99/41257 and WO 99/45930. Thecomplexes or assay systems, that comprise a formula 1 compound and oneor more of these molecules are embodiments of the invention, as are theuse of these compositions when employed in the practice of any of theassay methods or in any of the clinical treatment methods disclosedherein or in the cited references.

In exemplary applications, invention embodiments include a methodcomprising contacting a formula 1 compound(s) with a cell(s), wherebythe formula 1 compound(s) forms a complex with a steroid hormonereceptor or results in the modulation of a biological activity of thesteroid hormone receptor or a gene that it regulates. The steroidhormone receptor may be an orphan nuclear hormone receptor or acharacterized receptor such as the glucocorticoid receptor, estrogenreceptor or the androgen receptor that displays a moderate or highbinding affinity for the formula 1 compound(s). In some embodiments, thesteroid receptor is a known steroid receptor. Biological effects frominteraction of a formula 1 compound and a receptor can lead tointerference with the replication or development of a pathogen or thecell(s) itself. For example, expression of HIV transcripts inHIV-infected cells may be altered. The receptor-formula 1 compoundcomplex may directly interfere with LTR-dependent transcription of HIVgenes, leading to reduced viral replication. Alternatively, such effectscan include the decreased synthesis or biological activity of a proteinor gene product that is associated with the establishment, maintenanceor progression of a disease condition described herein or in the citedreferences.

Invention embodiments include compositions comprising a partiallypurified or a purified complex comprising a formula 1 compound and asteroid receptor. Such a steroid receptor(s) may be an orphan steroidreceptor or a characterized steroid receptor, where either type bindsthe formula 1 compound with a moderate or high binding affinity, e.g.,less than about 0.5-10×10⁻⁶M, usually less than about 1×10⁻⁷ M, or, forhigher affinity interactions, less than about 0.01-10×10⁻⁹ M. Theformula 1 compound(s) may also enhance immune responses such that bothimmune responses and altered intracellular conditions simultaneouslyexist to ameliorate one or more of the pathological conditions describedherein.

The formula 1 compounds may be used to identify receptors that modulatebiological responses, e.g., receptors that participate in effectingenhanced Th1 cytokine synthesis. Invention embodiments include a method,“Method 1”, which permits the determination of one or more effects of atest compound on a steroid receptor in various biological systems.Generally, the test compound is a formula 1 compound. Such systemsinclude cells containing a DNA construct that constitutively orinducibly expresses a steroid receptor(s) of interest, e.g., SXR, CARβ,RXR, PXR, PPARα, PPARβ, PPARγ or mixtures or dimers thereof, e.g.,SXR/RXR. In other biological systems, the steroid receptor can be underthe transcriptional control of a regulatable promoter. Alternatively,the expression another gene such as a steroid-inducible gene, e.g., asteroid-inducible cytochrome P-450. For this method, a source of steroidreceptors is generally combined with a means of monitoring them, e.g.,by measuring the transcription of a gene regulated by the receptor.Cells that comprise the steroid receptor and optional monitoring meansare sometimes referred to herein as the “biological system.” Sources ofsteroid receptors include cell lines and cell populations that normallyexpress the steroid receptor of interest and extracts obtained from suchcells. Another source for a useful biological system for purposes ofthis method is tissues from experimental animals that express thereceptor.

In one aspect, method 1 allows one to determine one or more effects of aformula 1 compound on a steroid receptor using a method that comprises(a) providing a biological system, e.g., a cell extract, cells ortissue, comprising cells having a plurality of steroid receptors thatcomprise monomers, homodimers or heterodimers that comprise a steroidreceptor, e.g., SXR, CAR-β, RXR, PPARα, PPARβ, PPARγ, PXR or dimers thatcomprise one or more of these; (b) activating or inhibiting theplurality of monomers, homodimers or heterodimers that comprise thesteroid receptor by contacting the cells with a steroid receptor (e.g.,SXR, CAR-β, RXR, PPARα, PPARβ, PPARγ or PXR) agonist or antagonist; (c)removing substantially all of the steroid receptor agonist or antagonistfrom the cells; (d) determining an activity of the plurality ofmonomers, homodimers or heterodimers that comprise the steroid receptorwhile in an activated state in the absence of agonist or antagonist; (e)exposing the cells to the test compound; (f) determining at least oneeffect of the test compound on the activity of the plurality ofmonomers, homodimers or heterodimers that comprise one or more of thesteroid receptors while they remain substantially free of agonist orantagonist; and (g) optionally classifying the test compound as anagonist or an antagonist of the steroid receptor, or a neutral compoundhaving little or no detectable effect.

The effects that method 1 can measure includes determining or measuringan effect on a gene whose expression is affected by the steroidreceptor. The gene could be a gene associated with a pathologicalcondition such as an infectious agent, an immune disorder such as aninflammation condition or a hyperproliferation condition disclosedherein or in the cited references.

Thus, another aspect of method 1, “method 1A”, is determining if acompound not previously known to be a modulator of protein biosynthesiscan transcriptionally modulate the expression of a gene that encodes aprotein associated with the maintenance or treatment of one or moresymptoms of a pathological condition (the “target gene” or “targetprotein”). This method comprises: (a) contacting an assay system thatcomprises eucaryotic cells or a suitable lysate with a formula 1compound, wherein the eucaryotic cells or suitable lysate comprises one,two or a plurality of steroid receptor proteins and optionally comprisesone or more coactivator proteins and a DNA construct comprisingoperatively linked sequences that comprise (i) a modulatabletranscriptional regulatory sequence(s) of the target gene, (ii) apromoter of the gene, and (iii) either the target gene's nucleic acidcoding protein region or a coding region for a suitable reporter gene,either of which is capable of expressing the DNA construct's codingregion, which is coupled to, and under the control of, the modulatabletranscriptional regulatory sequence(s) and the promoter, underconditions such that the formula 1 compound, if capable of acting as atranscriptional modulator of the gene encoding the protein of interest,causes a measurable or detectable signal (e.g., an increased ordecreased nucleic or protein expression level compared to a suitablecontrol); (b) quantitatively or qualitatively determining the amount ofthe signal so produced; and (c) optionally comparing the amount of thesignal so determined with the amount of signal detected in the presenceof the formula 1 compound with a suitable control, e.g. in the absenceof any compound being tested or upon contacting the sample with a knownactivator or inhibitor of expression of the target gene so as toidentify the chemical as one that causes a change in the detectablesignal produced by the polypeptide. The method thus permits one todetermine if the formula 1 compound specifically transcriptionallymodulates expression of the target gene or protein. Suitable steroidreceptors and their coactivators are as described herein, e.g., androgenreceptor, estrogen receptor, glucocorticoid receptor, mineralcorticoidreceptor, aldosterone receptor, PPARα, PPARβ, PPARγ, PPARδ, RXR or CARβ.Information regarding these receptors has been described, see, e.g.,relevant U.S. patents cited herein and PCT publication WO 0025800 and WO0031286.

In method 1A the assay system may comprise a transformed cell line,primary cells or untransformed cells or a suitable lysate or extract ofany of these. Exemplary cell lines include ones derived from human orother mammalian tumors or precancers. The source of such cells may behuman or animal, e.g., a mammal such as a primate or a rodent. In someembodiments, the assay system comprises a rodent such as a mouse,transgenic mouse or other transgenic animal (see e.g., PCT publicationWO 000602). Measurement of the formula 1 compound's effect on the assaysystem includes detecting increased or decreased expression of nucleicacid or polypeptide by the target gene in response to the formula 1compound's presence. Exemplary conditions include conducting the methodunder conditions suitable for maintaining the cells in tissue culture orunder conditions suitable for enzyme assays using cell extracts orlysates, e.g., tissue culture medium or a buffered aqueous solution thatis, e.g., a nearly isotonic solution at about 32-38° C., a pH of about 6to about 8, a formula 1 compound concentration of about 1×10⁻¹¹ M toabout 1×10⁻³ M (including any concentration in any single unit incrementof, e.g., 1×10⁻¹¹ M, e.g., 1×10⁻¹⁰ M, 1×10⁻⁹ M, 1×10⁻⁸ M, 1×10⁻⁷ M,1×10⁻⁶ M, 1×10⁻⁵ M or 1×10⁻⁴ M) and contacting the formula 1 compoundand the assay system for about 20 minutes to about 72 hours. Detectionof a change in expression of the target gene or the reporter geneincludes, e.g., detecting (1) changes in gene nucleic acid levels byqualitative or quantitative PCR methods and (2) changes in gene proteinlevels by enzyme or antibody-based assays such as measuring enzymeactivity using a suitable substrate or measuring protein levels, e.g.,by ELISA or western blotting.

In conducting method 1A, one typically contacts a sample that contains apredefined number of identical or essentially identical eucaryoticcells, e.g. about 5×10³ to about 5×10⁶ cells, with a predeterminedconcentration of a compound of formula 1. The eucaryotic cells comprisea DNA construct that is made using conventional molecular biologymethods and protocols. The assay system is maintained under conditionssuch that the formula 1 compound, if capable of acting as atranscriptional modulator of the gene encoding the protein of interest,causes a measurable or detectable signal to be produced by thepolypeptide expressed by the reporter gene. Once sufficient time forgeneration of a detectable response or signal has passed, one candetermine the amount of the signal produced. Typically the response orsignal is measured quantitatively, but a qualitative measurement can beuseful for rapid screening purposes.

For method 1A, one can also optionally compare the detectable signalwith the amount of produced signal that (i) one detects in the absenceof any formula 1 compound or (ii) when contacting the sample with otherchemicals, which identifies the formula 1 compound as a chemical thatcauses a change in the detectable signal the polypeptide produces. Onethen typically determines whether the formula 1 compound specificallytranscriptionally modulates expression of the gene associated with themaintenance or treatment of one or more symptoms of the pathologicalcondition.

Other aspects of the method 1 and 1A include a screening methodcomprising separately contacting each of a plurality of identical,essentially identical or different samples, each sample containing apredefined number of such cells with a with a predeterminedconcentration of each different formula 1 compound to be tested, e.g.,wherein the plurality of samples comprises more than about 1×10³ or morethan about 1×10⁴ samples or about 0.5-5×10⁵ samples. In other aspectsone determines the amount of RNA by quantitative polymerase chainreaction. In any of methods 1 or 1A, a formula 1 compound such as anyone of those described or named herein may be utilized.

Aspects of the invention include another method, “method 2”, whichcenters on identifying a gene whose expression is modulated by acandidate binding partner for infectious disease therapeutic agents.Typically the binding partner is a steroid receptor, e.g., a monomer,homodimer or heterodimer that comprises SXR, CAR-β, PXR, PPARα, PPARβ,PPARγ, PPARδ or RXR or a homolog or isoform thereof. The steroidreceptor is typically present as a complex that comprises, e.g., theformula 1 compound and the regulated gene's DNARS, which the steroidreceptor, or a complex that comprises the steroid receptor, recognizesand specifically binds to. Such complexes can also comprise atranscription factor that binds to the steroid receptor or to nucleicacid sequences adjacent to or near the DNARS. Exemplary transcriptionfactors that may be present include one or more of ARA54, ARA55, ARA70,SRC-1, NF-κB, NFAT, AP1, Ets, p300, CBP, p300/CBP, p300/CPB-associatedfactor, SWI/SNF and human homologs of SWI/SNF, CBP, SF-1, RIP140, GRIP1and Vpr. In general, one provides a first and a second group of cells invitro or in vivo and contacts the first group of cells with theinfectious disease therapeutic agent, but does not contact the secondgroup of cells in vitro or in vivo with the infectious diseasetherapeutic agent. Recovering RNA from the cells, or generating cDNAderived from the RNA, is accomplished by conventional protocols.Analysis of the RNA, or cDNA derived from the RNA, from the first andthe second group of cells identifies differences between them, which onecan use to identify a gene whose regulation is modulated by thecandidate binding partner for the infectious disease therapeutic agentor any DNARS associated with that gene.

An aspect of method 2 is determining the capacity of a formula 1compound to modulate, or participate in the modulation of, thetranscription of a gene associated with the maintenance or treatment ofone or more symptoms of a pathological condition. It is expected that ingeneral, the formula 1 compounds will cause an increase in thetranscription of such genes. The pathological condition is typically oneassociated with an infectious agent, e.g., virus, parasite or bacterium,but can also include an immune condition, e.g., an autoimmune conditionor an immune deficiency. The pathological condition may also be aninsufficient immune response to an infection or an insufficient responseto a hyperproliferation condition or malignancy. Other pathologicalconditions that one can apply the method to are inflammation conditions.

In some aspects, the formula 1 compounds used in method 2 will belabeled. Such compounds are prepared by conventional methods usingstandard labels, such as radiolabels, fluorescent labels or other labelsas described herein and in the cited references.

An embodiment of method 2 involves analyzing the RNA, or cDNA derivedfrom it, by subtraction hybridization. In this embodiment, the RNA orcDNA obtained from the first and second groups of cells is hybridizedand the resulting duplexes are removed. This allows recovery of nucleicacids that encode genes whose transcription is modulated by thecandidate binding partner, which is usually a steroid receptor. One canuse conventional methods to amplify and obtain nucleic acid and proteinsequence information from the nucleic acids recovered by this method.The nucleic acid sequences that are transcriptionally induced orrepressed by the formula 1 compound are candidate binding partners.

A transcriptionally induced gene(s) will be enriched in the group 1cells treated with the formula 1 compound, while any repressed gene(s)will be depleted or absent. In these embodiments, the RNA recoveredafter removal of duplexes is typically amplified by standard RT-PCR orPCR protocols. These protocols typically use specific sets of randomprimer pairs, followed by analysis of the amplified nucleic acids by gelelectrophoresis. Nucleic acids that are induced by the formula 1compound will appear as a band(s), usually duplex DNA, that is notpresent in the control or second set of cells. Nucleic acids that aretranscriptionally repressed by the formula 1 compound's binding partnerwill be depleted or absent in the first group of cells. Once such genecandidates are identified, they can be cloned and expressed and thecapacity of the DNARS associated with the gene to form a complex thatcomprises a candidate binding partner and an optionally labeled formula1 compound is analyzed by conventional methods, e.g., equilibriumdialysis, affinity chromatography using, e.g., the DNARS immobilized ona column, or coprecipitation of complexes that comprise an optionallylabeled DNARS and candidate binding partner using anti-binding partnerantibodies. Nucleic acid sequence analysis is usually used to identifyregions adjacent to the coding regions of the regulated gene to identifyany DNARS associated with the gene. The identity of a DNARS can beestablished by the binding to the DNARS of complexes that comprise acandidate binding partner, e.g., a steroid receptor, and optionally alsocomprise a formula 1 compound. The location and identity of the DNARScan be accomplished by DNA footprinting or other methods for detectingbinding interactions. The DNARS, the receptor or the formula 1 compoundcan be labeled in these variations of method 2.

In general, the second group of cells will be identical or essentiallyidentical to the first group of cells. In embodiments (for both methods1 and 2) where the cells are “essentially identical”, the first or thesecond group of cells may differ from each other by the presence orabsence of a DNA construct(s) that expresses (i) a steroid receptorand/or (ii) an easily detected protein, e.g., a β-galactosidase, aperoxidase, a phosphatase, a luciferase or a chloramphenicolacetyltransferase, whose transcriptional regulation is usually modulatedby a steroid receptor. In these embodiments, the difference between thefirst and the second group of cells is used to facilitate the analysisof the biological effects of the formula 1 compound and the steroidreceptor binding partner. Groups of cells are considered “identical” ifthey do not display known or obvious morphological or geneticdifferences.

Usually, the second group of cells will serve as a control, and theywill thus not be exposed to any formula 1 compound before obtaining theRNA or cDNA. But, for some embodiments, one can expose the second groupof cells to a known agonist or antagonist of the steroid receptorbinding partner. This allows one to compare the potency of the formula 1compound with the potency of the agonist or antagonist.

In other embodiments, one can modify method 2 by providing a third groupof cells, which is optionally used as an untreated control when thesecond group of cells is treated with a steroid receptor agonist orantagonist. In these embodiments, one will typically compare the effectof the formula 1 compound and the agonist or the antagonist of theexpression of a gene or DNA construct. The DNA construct would comprisea promoter or other regulatory sequences that are subject totranscriptional modulation, usually increase transcription, by theformula 1 compound in concert with its binding partner.

The formula 1 compounds can directly or indirectly modulate the activityor synthesis of one or more biological ligands to effect a detectablebiological response or activity change. To facilitate the identificationof candidate binding partners for the formula 1 compounds, one can use aradiolabeled formula 1 compound that is linked to a support, usually asolid support, as a means to recover the candidate binding partners. Theformula 1 compound can be linked to the support through a variable groupthat is bonded to the formula 1 compound, e.g., at the 2-, 3-, 7-, 11-,15-, 16- or 17-position of the steroid nucleus. Linking agents are knownfor such uses and include homobifunctional and heterobifunctionalagents, many of which are commercially available. The linker one useswill typically comprise about 2-20 linked atoms. The linked atomsusually comprise mostly carbon, with one, two or three oxygen, sulfur ornitrogen atoms that optionally replace one or more carbon or hydrogenatoms. One can use a cDNA expression library that one has made fromsuitable cells or tissues as a source of candidate binding partners. Thecells or tissues can be obtained from a mammalian or a vertebrate host,e.g., human, mouse, bird, primate, or from other sources, e.g., insects(e.g., Drosophila), other invertebrates (e.g., yeast, bacteria,Mycoplasma sp., Plasmodium sp., Tetrahymena sp., C. elegans) or otherorganism groups or species listed herein or in the cited references.Suitable tissues include skin, liver tissue or cells, includinghepatocytes and Küpfer cells, fibrocytes, monocytes, dendritic cells,kidney cells and tissues, brain or other central nervous system cells ortissues, including neurons, astrocytes and glial cells, peripheralnervous system tissues, lung, intestine, placenta, breast, ovary,testes, muscle, including heart or myocyte tissue or cells, white bloodcells, including T cells, B cells, bone marrow cells and tissues, lymphtissues or fluids and chondrocytes.

Typically a candidate binding partner that one isolates from a non-humansource will have a human homolog that has similar binding properties forthe formula 1 compound. Non-human candidate binding partners can thus beused to facilitate recovery of the human homologs, e.g., by preparingantiserum for precipitating the human homolog from a solution thatcomprises the human homolog or by comparing the sequence of thenon-human candidate binding partner with known human gene sequences.Once a source of the candidate binding partner is obtained, it can becontacted with labeled formula 1 compound, usually radiolabeled with,e.g., ¹⁴C or ³H, and complexes that comprise the labeled formula 1compound and the candidate binding partner is recovered using, e.g.,affinity chromatography or antibody precipitation methods. The recoveryof the complex provides a source of at least partially purifiedcandidate binding partner, i.e., the candidate binding partner isenriched, e.g., at least 10-fold enriched, or at least 100-foldenriched, or at least 500-fold enriched, compared to its abundance inthe original candidate binding partner source material.

Embodiments of the invention include a composition comprising apartially purified (purified at least about 2-fold to about 10-foldrelative to natural sources, e.g., cells or a cell lysate) complex or apurified (purified at least about 20-fold to about 5000-fold relative tonatural sources, e.g., cells or a cell lysate) complex (where thepartially purified or purified complex is optionally isolated)comprising a formula 1 compound and a steroid receptor, a serumsteroid-binding protein (e.g., human serum albumin, a1-acidglycoprotein, sex hormone-binding globulin, testosterone-bindingglobulin, corticosteroid-binding globulin, androgen binding protein(rat)) or another binding partner, e.g., transcription factor or DNARS.An aspect of these compositions includes a product produced by theprocess of contacting the partially purified or the purified compositionwith one or more cells, one or more tissues, plasma or blood.

Other embodiments include a method to modulate a cellular response or todetermine a biological activity of a formula 1 compound comprising: (a)contacting the formula 1 compound(s) with a cell or cell population; (b)measuring one or more of (i) a complex between a binding partner and theformula 1 compound, (ii) proliferation of the cell or cell population,(iii) differentiation of the cell or cell population (iv) an activity ofa protein kinase C, (v) a level of phosphorylation of a protein kinase Csubstrate, (vi) transcription of one or more target genes, (vii)inhibition of unwanted cellular responses to certain steroids, e.g.,inhibition of glucocorticoid-induced immune suppression or inhibition ofglucocorticoid-induced bone loss, (viii) inhibition or modulation ofsteroid-induced transcription, e.g., increased or decreased expressioninduced by glucocorticoids or sex steroids or (ix) inhibition of HIVLTR-driven transcription; and (c) optionally comparing the resultobtained in step (b) with an appropriate control. Aspects of thisembodiment include (i) the method wherein the binding partner is asteroid receptor, a transcription factor or a DNARS, (ii) the methodwherein the biological activity determined is a modulating activity ofthe formula 1 compound for replication or cytopathic effects associatedwith a retrovirus, a hepatitis virus or a protozoan parasite, (iii) themethod wherein the biological activity determined is a modulatingactivity of the formula 1 compound for replication, cytopathic effectsassociated with the retrovirus, the hepatitis virus or the protozoanparasite or the biological activity determined is metabolism (assay by³H-thymidine uptake or other assay as referenced or described herein) ofa cell or cell population comprising NK cells, phagocytes, monocytes,macrophages, basophils, eosinophils, dendritic cells, synoviocytes,microglial cells, fibrocytes, transformed (neoplastic) cells,virus-infected cells, bacteria-infected cells or parasite-infectedcells, and (iv) the method wherein the target gene is a virus gene, abacterial gene, a parasite gene, a gene associated with cancer, e.g.,wherein the virus gene is a DNA or an RNA polymerase gene, a reversetranscriptase gene, an envelope gene, a protease gene or a geneassociated with viral nucleic acid replication or a viral structuralgene.

Another embodiment is a method comprising contacting a complex thatcomprises a steroid receptor and a formula 1 compound with atransactivator protein, whereby a complex comprising the steroidreceptor protein, the formula 1 compound and the transactivator proteinforms, wherein the transactivator protein is in (1) a cell or tissueextract (e.g., nuclei, lysate containing nuclei or lysate without nucleifrom a cell(s) or tissue(s)), (2) a partially purified or purified cellor tissue extract, (3) a cell(s) in tissue culture or (4) a cell(s) in asubject, where any of (1)-(4) optionally comprises a target gene (nativegene or introduced by standard gene manipulation techniques) whose levelof expression is optionally assayed after the complex forms. In some ofthese embodiments, the transactivator protein is partially purified orpurified and is in the cell or tissue extract or the partially purifiedor purified cell or tissue extract. The transactivator protein may beTIF-1, CBP/P300, TRIP1/SUG-1, RIP-140, SRC1α/P160, or TIF-2/GRIP-1. Inany of these embodiments the complex comprising the steroid receptorprotein, the formula 1 compound and the transactivator protein mayincrease or decrease transcription of the target gene compared to asuitable control (e.g., control under same conditions, but lacking anyadded compound that corresponds to the formula 1 compound, or whereanother compound (e.g., a steroid that is known to bind to the steroidreceptor) is used as a benchmark or reference standard against whichaltered target gene expression is measured). In these methods, thetarget gene may be a pathogen gene (e.g., virus, bacterium, parasite,fungus, yeast) or a gene associated with a pathological condition(autoimmunity, inflammation, hyperproliferation).

The formula 1 compounds are suitable for use in certain describedmethods that use steroids to modulate biological activities in cells ortissues. For example, a formula 1 compound(s) can be used to selectivelyinteract with specific steroid receptors or steroid orphan receptor, ortheir subtypes, that are associated with a pathological condition(s) ina subject, essentially as described in U.S. Pat. No. 5,668,175. In theseapplications, the formula 1 compound may act as a ligand for thereceptor to modulate abnormal expression of a gene product(s) thatcorrelates with the pathological condition (a steroid hormone responsivedisease state). Such genes are normally regulated by steroid hormones.In other applications, one can use the formula 1 compounds to screen forligands that bind to or detectably affect a biological activity of asteroid receptor or steroid orphan receptor and one or moretranscription factors (or cofactors) such as AP-1 and/or with a DNAsequence(s), essentially as described in U.S. Pat. No. 5,643,720.Similarly, the formula 1 compounds can be used essentially as describedin U.S. Pat. Nos. 5,597,693, 5,639,598, 5,780,220, 5,863,733 and5,869,337 to detectably modulate a biological activity of one of thesemolecules. In some of these embodiments, the formula 1 compound(s) islabeled to facilitate its use. Suitable labels are known in the art andinclude radiolabels (e.g., ³H, ¹⁴C, ³²P, ³⁵S, ¹³¹I, ⁹⁹Tc and otherhalogen isotopes), fluorescent moieties (e.g., fluorescein, resorufin,Texas Red, rhodamine, BODIPY, arylsulfonate cyanines), chemiluminescentmoieties (e.g., acridinium esters), metal chelators, biotin, avadin,peptide tags (e.g., histidine hexamer, a peptide recognized bymonoclonal or polyclonal antibodies), covalent crosslinking moieties.One prepares the labeled compounds according to known methods.

Methods suitable to measure the cellular response or biological effectscaused by various compounds, e.g., activation, on immune system cells(e.g., NK cells, phagocytes, monocytes, macrophage, neutrophils,eosinophils, dendritic cells, synoviocytes, microglial cells,fibrocytes) have been described, e.g., Jakob et al., J. Immunol. 1998161:3042-3049, Pierson et al., Blood 1996 87:180-189, Cash et al., Clin.Exp. Immunol. 1994 98:313-318, Monick et al., J. Immunol. 1999162:3005-3012, Rosen et al., Infect. Immun. 1999 67:1180-1186, Grunfeldet al., J. Lipid Res. 1999 40:245-252, Singh et al., Immunol. Cell Biol.1998 76:513-519, Chesney et al., Proc. Natl. Acad. Sci. USA 199794:6307-6312, Verhasselt et al., J. Immunol. 1999 162:2569-2574, Aviceet al., J. Immunol. 1999 162:2748-2753, Cella et al., J. Exp. Med. 1999189:821-829, Rutalt et al., Free Radical Biol. Med. 1999 26:232-238,Akbari et al., J. Exp. Med. 1999 189:169-178, Hryhorenko et al.,Immunopharmacology 1998 40:231-240, Fernvik et al., Inflamm. Res. 199948:28-35, Cooper et al., J. Infect. Dis. 1999 179:738-742, Betsuyaku etal., J. Clin. Invest. 1999 103:825-832, Brown et al., Toxicol. Sci. 199846:308-316, Sibelius et al., Infect. Immunol. 1999 67:1125-1130. The useof formula 1 compounds in such methods are aspects of the invention andthey permit, e.g., measurement of the biological effects of formula 1compounds on, e.g., one or more of (1) the cell's biological activities,(2) genes whose expression is regulated by the formula 1 compound or (3)a steroid receptor. Exemplary biological effects that the formula 1compounds may exert include one or more of (1) stimulation of ion fluxor ion channel activity in one or more immune cell subsets such as oneor more of those described herein, (2) binding to one or more ligandssuch as a steroid receptor and modulation of a biological activity ofthe receptor, (3) detectably enhanced transcription of one or more geneswhose expression is affected by a steroid receptor(s) or otherbiomolecule whose activity is directly or indirectly affected by theformula 1 compound's presence and (4) detectably decreased transcriptionof one or more genes whose expression is affected by a steroidreceptor(s) or other biomolecule whose activity is directly orindirectly affected by the formula 1 compound's presence.

Embodiments include any of the methods described above, e.g., method 1,wherein the cells or biological system comprises NK cells, phagocytes,monocytes, macrophage, neutrophils, eosinophils, dendritic cells,synoviocytes, microglial cells, glial cells, fibrocytes or hepatocytes,that optionally comprise a DNA construct that expresses one or twocloned steroid receptors. The method optionally analyzes the effect of aformula 1 compound on the cells compared to controls. Controls includethe use of a known agonist or antagonist for the steroid receptor or thecomparison of cells exposed to a formula 1 compound with control cells(usually the same cell type as the treated cells) that are not exposedto the formula 1 compound. A response, e.g., activation of the steroidreceptor can be measured by known assays compared to controls.

The formula 1 compound will, in some cases modulate (increase ordecrease) transcription of one or more genes in the cells. In othercases, the formula 1 compound will enhance lysosome movement in one ormore of the subject's NK cells, phagocytes, monocytes, macrophages,neutrophils, eosinophils, dendritic cells synoviocytes, microglial cellsor fibrocytes. Such effects will typically be mediated directly orindirectly through one or more transcription factors or steroidreceptors that act to modulate gene transcription, e.g., cause enhancedprotein kinase C (a PKC such as PKCα, PKCβ, PKCγ or PKCζ) activity inthe cells used in the assay, or another effect as disclosed herein.

Other related embodiments are a composition comprising a partiallypurified or a purified complex comprising a formula 1 compound and asteroid receptor, a serum steroid-binding protein (e.g., human serumalbumin, α1-acid glycoprotein, sex hormone-binding globulin,testosterone-binding globulin, corticosteroid-binding globulin, androgenbinding protein (rat) or a homolog or isoform of any of these) oranother binding partner, e.g., transcription factor or DNARS. An aspectof these compositions includes a product produced by the process ofcontacting the partially purified or the purified composition with oneor more cells, one or more tissues, plasma or blood.

In a related embodiment, a formula 1 compound is used to exert acytostatic effect on a subject's cells, e.g., mammalian cells, in vitroor in vivo. Typically such cells are lymphoid cells, e.g., T cellpopulations from, e.g., blood or organs that are rich in lymphoid cells(e.g., spleen, lymph tissue or nodes), or transformed T cell lines. Suchactivity provides an estimate of the potency of formula 1 compounds tomediate immunological effects, such as enhancing Th1 immune responses orsuppressing expression of one or more Th2-associated cytokines. Thus, aninvention method comprises (a) contacting a formula 1 compound andlymphoid cells in vitro, (b) determining the degree of cytostasis thatthe compound exerts to identify a cytostatic compound and (c) optionallyadministering the cytostatic compound to an immune suppressed subject todetermine the effect of the compound on one or more of the subject'simmune responses as described herein, e.g., enhanced Th1 cytokine orcell response or decreased Th2-associated cytokine expression.Typically, such methods are conducted using a range of formula 1compound concentrations and suitable controls, such as a knowncytostatic agent or a blank that contains solvent that lacks the formula1 compound. Inhibition of cell proliferation is measured by standardmethods. Methods to measure the cytostatic effects of the compoundsincludes measuring viable cell numbers in treated and untreated culturesor by measuring DNA synthesis using e.g., ³H-thymidine incorporationinto DNA in treated and untreated cultures. Typical ranges of formula 1concentrations in the cell growth medium are about 0.1 μM to about 100μM, using about 4-6 different concentrations of compounds with a fixednumber of cells (e.g., about 0.4×10⁵ to about 5×10⁵). The formula 1compound is left in contact with the cells in tissue culture for asufficient time to observe cytostasis, e.g., about 16 hours to about 6days, typically about 24-72 hours. In these embodiments, one mayoptionally screen for modulation of a biological activity of a steroidreceptor, e.g., activation of PPARα, which may be associated with thecytostasis the compound induced.

Other therapeutic and biological applications and activities. Theformula 1 compounds are useful to treat autoimmune or metabolicconditions or disorders, or their symptoms, in subjects such as mammalsor humans that relate to impaired insulin synthesis or use or thatrelate to abnormal or pathological lipid or cholesterol metabolism orlevels. Such conditions and symptoms include Type 1 diabetes (includingImmune-Mediated Diabetes Mellitus and Idiopathic Diabetes Mellitus),Type 2 diabetes (including forms with (1) predominant or profoundinsulin resistance, (2) predominant insulin deficiency and some insulinresistance and (3) forms intermediate between these), obesity,hyperglycemia, hyperlipidemia conditions such as hypertriglyceridemiaand hypercholesterolemia. In diabetes, the compounds are useful to (1)enhance β-cell function in the islets of Langerhans (e.g., increaseinsulin secretion), (2) reduce the rate of islet cell damage, (3)increase insulin receptor levels or activity to increase cellsensitivity to insulin and/or (4) modulate glucocorticoid receptoractivity to decrease insulin resistance in cells that are insulinresistant. The compounds are thus useful to treat, prevent, ameliorateor slow the progression of diabetes or hyperglycemia, or a relatedsymptom or condition, in a subject such as a human or a mammal.

Beneficial effects that can the formula 1 compounds can exert on suchrelated symptoms or conditions include improved glucose tolerance,improved glucose utilization, decreased vascular disease (e.g.,decreased severity or progression of microvascular or macrovasculardisease, including nephropathy, neuropathy, retinopathy, hypertension,cerebrovascular disease and coronary heart disease), decreased severityor progression of atherosclerosis, decreased severity or progression ofan arteriosclerosis condition (e.g., coronary arteriosclerosis,hyperplastic arteriosclerosis, peripheral arteriosclerosis orhypertensive arteriosclerosis), decreased level or activity ofinflammatory macrophages (foam cells) in atherosclerotic plaques,decreased severity or progression of diabetic osteoarthropathy,decreased severity or progression of skin lesions, decreased severity orprogression of ketosis, decreased generation of autoantibodies againstislet cells or decreased expression or levels of one or more of IL-1(e.g., IL-1β), IL-6, TNF (e.g., TNFα), and IFN-γ. In these any of thesediseases or conditions, the formula 1 compounds can also modulate, e.g.,enhance CARβ, RXR, PPARα or PPARβ levels. As used herein, obesityincludes a human with a body mass index of about 27, 28, 29, 30, 31, 32,33, 34 or greater.

The formula 1 compounds are useful in treating insulin resistance andassociated symptoms and conditions. Insulin resistance is typicallyobserved as a diminished ability of insulin to exert its biologicalaction across a broad range of concentrations. This leads to less thanthe expected biologic effect for a given level of insulin. Insulinresistant subjects or human have a diminished ability to properlymetabolize glucose or fatty acids and respond poorly, if at all, toinsulin therapy. Manifestations of insulin resistance includeinsufficient insulin activation of glucose uptake, oxidation and storagein muscle and inadequate insulin repression of lipolysis in adiposetissue and of glucose production and secretion in liver. Insulinresistance can cause or contribute to polycystic ovarian syndrome,impaired glucose tolerance, gestational diabetes, hypertension, obesity,atherosclerosis and a variety of other disorders. Insulin resistantindividuals can progress to a diabetic state. The compounds can also beused in the treatment or amelioration of one or more conditionassociated with insulin resistance or glucose intolerance including anincrease in plasma triglycerides and a decrease in high-densitylipoprotein cholesterol, high blood pressure, hyperuricemia, smallerdenser low-density lipoprotein particles, and higher circulating levelsof plasminogen activator inhibitor-1. Such diseases and symptoms havebeen described, see, e.g., G. M. Reaven, J. Basic Clin. Phys. Pharm.1998, 9: 387-406, G. M. Reaven, Physiol. Rev. 1995, 75: 473-486 and J.Flier, J. Ann. Rev. Med. 1983, 34:145-60.

The compounds can thus be used in diabetes, obesity, hyperlipidemia orhypercholesterolemia conditions to reduce body fat mass, increase musclemass or to lower one or more of serum or blood low density lipoprotein,triglyceride, cholesterol, apolipoprotein B, free fatty acid or very lowdensity lipoprotein compared to a subject that would otherwise beconsidered normal for one or more of these characteristics. Thesebeneficial effects are typically obtained with little or no effect onserum or blood high density lipoprotein levels. The formula 1 compoundsare useful to reduce or slow the rate of myocardial tissue or myocytedamage, e.g., fibrosis, or to enhance cardiac fatty acid metabolism inconditions, such as inflammation, where fatty acid metabolism isdepressed or decreased. Elevated cholesterol levels are often associatedwith a number of other disease states, including coronary arterydisease, angina pectoris, carotid artery disease, strokes, cerebralarteriosclerosis, and xanthoma, which the formula 1 compounds canameliorate or slow the progression or severity of. Abnormal lipid andcholseterol conditions that can be treated include exogenoushypertriglyceridemia, familial hypercholesterolemia, polygenichypercholesterolemia, biliary cirrhosis, familial combinedhyperlipidemia, dysbetalipoproteinemia, endogenous hypertriglyceridemia,mixed hypertriglyceridemia and hyperlipidemia or hypertriglycidemiasecondary to alcohol consumption, diabetic lipemia, nephrosis or drugtreatments, e.g., corticosteroid, estrogen, colestipol, cholestyramineor retinoid treatments. Dosages, routes of administration and dosingprotocols for the formula 1 compounds are essenitally as describedherein. Where the condition is chronic, the formula 1 compounds willgenerally be administered to a subject such as a human for a relativelylong time period, e.g., for about 3 months to about 10 years or more.Dosages, routes of administration and dosing protocols for the formula 1compounds are essenitally as described herein. Dosing of the compoundcan be daily or intermittent using a dosing protocol using dosages asdescribed herein, e.g., about 0.01 to about 20 mg/kg of a formula 1compound administered to a subject once or twice per day daily orintermittently. The use of the formula 1 compounds can be combined withother suitable treatments, e.g., diet control or HMG-CoA reductaseinhibitors such as Simvastatin™, Pravastatin™, Mevastatin™ orLovastatin™.

The formula 1 compounds are also useful for preventing, slowing theprogression of or treating certain chronic conditions in a subject suchas a mammal or a human. Chronic conditions include diseases andconditions that arise or develop over a relatively long time period,e.g., over about 3 months to 10 years or more. Such conditions includechronic renal failure, which may result from polycystic kidney disease,from, e.g., an autoimmune condition such as acute or chronicglomerulonephritis, or from diabetes, interstitial nephritis,hypertension and other conditions discussed elsewhere herein. Chronicconditions include chronic pulmonary conditions such as chronicbronchitis, lung fibrosis, right ventricular hypertrophy, pulmonaryhypertension, emphysema, asthma and chronic obstructive pulmonarydisease, which may be treated with a formula 1 compound. Theseconditions or their symptoms may be mild, moderate or severe. Thesubject may be suffering from the disease or condition or may be subjectto developing the condition, e.g., the subject may display early signsor a predisposition to develop a chronic condition. Such treatment willgenerally facilitate prevention of the disease, delay the onset orseverity of the disease or condition, ameliorate one or more symptoms,e.g., reduce shortness of breath, coughing or dyspnea, or slowprogression of the disease or condition. In these and other chronicconditions described herein, the formula 1 compounds will generally beadministered to a subject such as a human for a relatively long timeperiod, e.g., for at least about 3 months to about 10 years or more.Dosages, routes of administration and dosing protocols for the formula 1compounds are essenitally as described herein. Dosing of the compoundcan be daily or intermittent using a dosing protocol using dosages asdescribed herein, e.g., about 0.1 to about 20 mg/kg of a formula 1compound administered to a subject once or twice per day daily orintermittently. The use of the formula 1 compounds can be combined withother treatments, e.g., β-agonists such as metaproterenol or albuterol,or corticosteroids, e.g., prednisone, for asthma or chronic obstructivepulmonary disease.

The formula 1 compounds can modulate the biological activity ofcytokines or interleukins that are associated with various immunedeficiency or dysregulation conditions, which may be transient orchronic. They can thus be used to ameliorate, treat or prevent naturallyoccurring age-related decline in immune function in a subject or immunedeficiency or dysregulation resulting from trauma, stress, burns,surgery, autoimmunity or infections as described herein. Such immunedeficiency dysregulation may be associated with, e.g., an age-relatedincrease in production of one or more of IL-4, IL-5 and IL-6 or anage-related decrease in production of one or more of IL-2, IL-3, γ-IFN,GM-CSF or antibodies. In these embodiments, the formula 1 compound isadministered to the subject to detectably decrease production or levelsof one or more of IL-4, IL-5 and IL-6 or to detectably increaseproduction or levels of one or more of IL-2, IL-3, IL-5, IL-12, GM-CSFand γ-IFN. These cytokine changes facilitate normalization of thesubject's immune responses. Such normalization can be observed byvarious means. These means include monitoring appropriate cytokine RNAor protein level(s) in the subject or by measuring biological responsessuch as restoration or detectable improvement of contacthypersensitivity in a subject with depressed or suboptimal contacthypersensitivity response. The formula 1 compounds can thus be used toenhance or restore a deficient or suboptimal immune response such ascontact hypersensitivity response in a subject with a chronic ortransient state of immune deficiency or dysregulation. In theseembodiments, the formula 1 compound is administered using the dosages,routes of administration and dosing protocols for the formula 1compounds essenitally as described herein. Treatment with the formula 1compounds is optionally combined with other appropriate treatments ortherapies essenitally as described herein, e.g., a antibacterial orantiviral agent(s) is coadministered with a formula 1 compound to treat,prevent or ameliorate an infection in an infected subject or a subjectsuffering from, e.g., a burn. Methods to measure changes in cytokinelevels or contact hypersensitivity are known and can optionally beapplied in these embodiments, see, e.g., U.S. Pat. Nos. 5,919,465,5,837,269, 5,827,841, 5,478,566.

The capacity of the formula 1 compounds to modulate immune functionspermits their use for treating, preventing, slowing the progression ofor alleviating the a symptom(s) of subjects with psychologicaldisorders, metabolic disorders, chronic stress, sleep disorders,conditions associated with sexual senescence, aging, or premature aging.Metabolic disorders include parathyroidism, pseudoparathyroidism,hypoparathyroidism, hypercalcemia, hypocalcemia and detectable symptomsthereof such as fatigue, constipation, kidney stones and kidneymalfunction. Chronic stress and related disorders include fibromyalgia,chronic fatigue syndrome and hypothalamic-pituitary axis dysregulation.In these embodiments, treatment of subjects with a formula 1 compound isoptionally combined with other suitable agents such as triiodothyronine,tetraiodothyronine, an insulin-like growth factor or insulin-like growthfactor binding protein-3.

Another aspect of the invention provides for the use of a formula 1compound and a flavonoid, e.g., a naragin flavonoid, to enhance thebioavailability of the formula 1 compound. In these embodiments, the aneffective amount of a flavonoid is administered to a subject who isreceiving a formula 1 compound. Typically about 1-10 mg of flavonoid perkg of body weight is administered to the subject a flavonoid such asbavachinin A, didymin (isosakuranetin-7-rutinoside or neoponcirin),flavanomarein (isookanine-7-glucoside), flavanone azine, flavanonediacetylhydrazone, flavanone hydrazone, silybin, cirsiliol,silychristin, isosilybin or silandrin. The flavonoid compound istypically administered with the formula 1 compound or a few hours, e.g.,about 1, 2 or 3 hours, before the formula 1 compound is administered tothe subject.

As noted above, in some embodiments a treatment with a formula 1compound is combined with a corticosteroid or glucocorticoid.Corticosteroids are used in a number of clinical situations to, e.g.,decrease the intensity or frequency of flares or episodes ofinflammation or autoimmune reactions in conditions such as acute orchronic rheumatoid arthritis, acute or chronic osteoarthritis,ulcerative colitis, acute or chronic asthma, bronchial asthma,psoriasis, systemic lupus erythematosus, hepatitis, pulmonary fibrosis,type I diabetes, type II diabetes or cachexia. However, manycorticosteroids have significant side effects or toxicities that canlimit their use or efficacy. The formula 1 compounds are useful tocounteract such side effects or toxicities without negating all of thedesired therapeutic capacity of the corticosteroid. This allows thecontinued use, or a modified dosage of the corticosteroid, e.g., anincreased dosage, without an intensification of the side effects ortoxicities or a decreased corticosteroid dosage. The side-effects ortoxicities that can be treated, prevented, ameliorated or reducedinclude one or more of bone loss, reduced bone growth, enhanced boneresorption, osteoporosis, immunosuppression, increased susceptibility toinfection, mood or personality changes, depression, headache, vertigo,high blood pressure or hypertension, muscle weakness, fatigue, nausea,malaise, peptic ulcers, pancreatitis, thin or fragile skin, growthsuppression in children or preadult subjects, thromboembolism,cataracts, and edema. Dosages, routes of administration and dosingprotocols for the formula 1 compound would be essentially as describedherein. An exemplary dose of formula 1 compound of about 0.5 to about 20mg/kg/day is administered during the period during which acorticosteroid is administered and optionally over a period of about 1week to about 6 months or more after dosing with the corticosteroid hasended. The corticosteroids are administered essentially using knowndosages, routes of administration and dosing protocols, see, e.g.,Physicians Desk Reference 54^(th) edition, 2000, pages 323-2781, ISBN1-56363-330-2, Medical Economics Co., Inc., Montvale, N.J. However, thedosage of the corticosteroid may optionally be adjusted, e.g., increasedabout 10% to about 300% above the normal dosage, without a correspondingincrease in all of the side effects or toxicities associated with thecorticosteroid. Such increases would be made incrementally over asufficient time period and as appropriate for the subject's clinicalcondition, e.g., daily corticosteroid dose increases of about 10% toabout 20% to a maximum of about 300% over about 2 weeks to about 1 year.

Such corticosteroids include hydrocortisone (cortisol), corticosterone,aldosterone, ACTH, triamcinolone and derivatives such as triamcinolonediacetate, triamcinolone hexacetonide, and triamcinolone acetonide,betamethasone and derivatives such as betamethasone dipropionate,betamethasone benzoate, betamethasone sodium phosphate, betamethasoneacetate, and betamethasone valerate, flunisolide, prednisone,fluocinolone and derivatives such as fluocinolone acetonide, diflorasoneand derivatives such as diflorasone diacetate, halcinonide,dexamethasone and derivatives such as dexamethasone dipropionate anddexamethasone valerate, desoximetasone (desoxymethasone), diflucortoloneand derivatives such as diflucortolone valerate), flucloroloneacetonide, fluocinonide, fluocortolone, fluprednidene, flurandrenolide,clobetasol, clobetasone and derivatives such as clobetasone butyrate,alclometasone, flumethasone, and fluocortolone.

In some applications, the formula 1 compound(s) may directly and/orindirectly interfere with replication, development or cell to celltransmission of a pathogen such as a virus or a parasite (malaria).Improvement in a subject's clinical condition may arise from a directeffect on an infectious agent or on a malignant cell. Interference withcellular replication can arise from inhibition of one or more enzymesthat a parasite or an infected cell uses for normal replication ormetabolism, e.g., glucose-6-phosphate dehydrogenase, which affectscellular generation of NADPH (see, e.g., Raineri et al., Biochemistry1970 9: 2233-2243). This effect may contribute to cytostatic effectsthat some formula 1 compounds can have. Modulation of cellular enzymesexpression or activity may also interfere with replication ordevelopment of a pathogen, e.g., HIV or malaria parasites or withreplication or development of neoplastic cells, e.g., inhibition ofangiogenesis. Clinical improvement will also generally result from anenhanced immune response such as an improved Th1 response.

Administration of a formula 1 compound can lead to a decrease inadenosine levels in a subject's tissue(s), e.g., lung or central nervoussystem tissue. This effect can be used to treat, prevent, ameliorate oneor more symptoms of or slow the progression of a disease(s) or clinicalcondition(s) where a relatively high level of adenosine is a factor inor can contribute to the disease or condition, e.g., in asthma.

Adenosine is associated with the symptoms of bronchial asthma, where itcan induce bronchoconstriction or contraction of airway smooth muscle inasthmatic subjects, see, e.g., J. Thorne and K. Broadley, AmericanJournal of Respiratory & Critical Care Medicine 149(2 pt. 1):392-3991994, S. Ali et al., Agents & Actions 37:165-167 1992, Bjorck et al.,American Review of Respiratory Disease 145:1087-1091 1992. This effectis not observed in non-asthmatic subjects. In the central nervoussystem, adenosine can inhibit the release of neurotransmitters such asacetylcholine, noradrenaline, dopamine, serotonin, glutamate, and GABA.It can also depress neurotransmission, reduce neuronal firing to inducespinal analgesia and it possesses anxiolytic properties, see, e.g., A.Pelleg and R. Porter, Pharmacotherapy 10:157 1990. In the heart,adenosine suppresses pacemaker activity, slows AV conduction, possessesantiarrhythmic and arrhythmogenic effects, modulates autonomic controland triggers the synthesis and release of prostaglandins. In addition,adenosine has vasodilatory effects and can modulate vascular tone.

The unwanted effects of excess adenosine can be ameliorated or reducedby administering sufficient amounts of a formula 1 compound to a subjectwho is subject to developing or who has an unwanted level of adenosinein one or more tissues or organs. In typical embodiments, one willadminister about 10 mg/kg/day to about 100 mg/kg/day of a formula 1compound to a subject over a period of about 1 week to about 4 months toeffect detectable changes in adenosine levels or amelioration in one ormore symptoms associated with high adenosine in one or more of thesubject's tissues. Such changes may be determined by comparing thesubject's adenosine levels before treatment with the formula 1 compoundis started. Alternatively, for subjects with symptoms that areconsistent with high adenosine levels, the decrease can be inferred bycomparing the normal level of adenosine in the target tissue(s) forsubjects of the same species and similar age or sex with the level thatis observed after treatment. Methods to measure adenosine levels inmammalian tissue are known and can optionally be used in theseembodiments, e.g., U.S. Pat. No. 6,087,351.

In some clinical conditions, the formula 1 compounds can inhibitactivated T lymphocytes in vivo, and they can inhibit the expression orbiological activity of one or more of TNF-α, IFN-γ, IL-6, IL-8 orinsulin like growth factor-1 receptor (IGF-1R) or IL-6 receptor. Thecompounds are thus useful to treat, prevent or ameliorate conditionswhere this is a component of pathology. Such conditions includeinflammation conditions such as psoriasis, psoriatic arthritis,osteoarthritis, and rheumatoid arthritis. The compound can thusameliorate the inflammation, e.g., by inhibiting expression of one ormore of TNF-α, IFN-γ, IL-6, IL-8 or IGF-1R. Also, the compounds caninhibit unwanted T cell activity. They can thus ameliorate one or morepsoriasis symptoms such as skin scaling, skin thickening, keratinocytehyperproliferation, deficient filaggrin expression (B. Baker et al., Br.J. Dermatol. 1984, 111:702), deficient strateum corneum lipid depositionor they can improve a clinical assessment such as the Psoriasis Activityand Severity Index. The formula 1 compounds can be delivered to asubject with psoriasis using topical or systemic formulations asdescribed herein. Topical formulations include gels, lotions and creams,e.g., as described herein. Daily or intermittent administration of thecompound can be used essentially as described herein. The use of theformula 1 compounds is optionally combined with one more currentpsoriasis treatments, e.g., topical emollients or moisturizers, tars,anthralins, systemic or topical corticosteroids, vitamin D analogs suchas calcitriol, methotrexate, etretinate, acitretin, cyclosporin, FK 506,sulfasalazine, ultraviolet B radiation optionally combined with one ormore of a topical corticosteroid, tar, anthralin, emollient ormoisturizer or ultraviolet A plus psoralen. Such additional treatmentsessentially would use known dosages and routes of administration, whichare applied, e.g., within a month before, during or within a month aftera treatment course with a formula 1 compound.

Other desirable modulation effects of the formula 1 compounds on cellsor tissues include (1) inhibition of one or more of bone resorption orcalcium release or gp80, gp130, tumor necrosis factor (TNF), osteoclastdifferentiation factor (RANKL/ODF), RANKL/ODF receptor, IL-6 or IL-6receptor expression or biological activity in, e.g., bone loss orosteoporosis conditions or in osteoclasts, or in cancers such asprostate cancer, metastatic breast cancer or metastatic lung cancer(e.g., with bone metastases), (2) inhibition of osteoclastogenesis orosteoclast development from progenitor cells, (3) enhancement of NFκBinhibition that is mediated by steroid receptors, e.g., enhancedinhibition of estrogen receptor-α or estrogen receptor-β mediatedinhibition of NFκB in inflammation, rheumatoid arthritis orosteoporosis, (4) enhancement of osteoblastogenesis, osteoblast, bonecallus or bone development, e.g., from progenitor cells in bonefractures, depressed bone healing situations (e.g., in a burn patient orin a patient being treated with a glucocorticoid), bone growth orosteoporosis or other bone loss conditions, by, e.g., modulation orenhancement of osteoblast replication or development or modulation orenhancement of the synthesis or biological activity of a transcriptionfactor such as Cbfa1, RUNX2 or AML-3 (5) normalization ofhypothalamic-pituitary-adrenal axis function in conditions where thereis dysregulation such as in chronic inflammatory diseases, chronicasthma or rheumatoid arthritis (increased cortisol to ACTH ratio), (6)modulation of ligand-gated ion channels in neurons in, e.g., depression,sleep or memory disorders, (8) modulation of G-protein coupled receptorsin neurons in, e.g., depression, sleep or memory disorders, (9)modulation, e.g., induction, of the synthesis or biological activity ofmetabolic enzymes such as a cytochrome (e.g., a CYP enzyme such asCYP1A1, CYP2B1, CYP2b10, CYP4A, CYP7A, CYP7A1, CYP7B, CYP7B1, P450 3A4,P450c17, P450scc, P450c21 or an isozyme, homolog or mutant of any ofthese) in cells or tissues such as liver cells, neurons, neuronprecursor cells, brain, breast, testes or colon, (10) enhancement ofcollagen synthesis or levels in, e.g., skin in aging or skin damagefrom, e.g., trauma, thermal injury or solar radiation, (11) inhibitionof nitric oxide production in cells or tissue, e.g., in nervous systemtissue or in microglial cells in dementias such as Alzheimer's disease,(12) enhancing glucose-stimulated insulin synthesis in hyperglycemia ordiabetes conditions, (13) modulation of gamma-aminobutyric acid (GABA),dopamine or N-methyl-D-aspartate (NMDA) receptor activity or levels in,e.g., brain tissue or neurons, (e.g., decreased GABA-mediated chloridecurrents or potentiation of neuronal response to NMDA in thehippocampus) in, e.g., conditions such as a dementia (Alzheimer'sDisease), depression, anxiety, schizophrenia or memory loss due to,e.g., aging or another condition described herein, (14) modulating(e.g., enhancing) the expression or activity of a transcriptionfactor(s), or a homolog(s) or isoform(s), such as SET, nerve growthfactor inducible protein B, StF-IT, SF-1 in cells or tissues such asnerve cells, neuronal precursor cells or liver cells, (15) inhibition ofeosinophil infiltration or reduction IgE levels in allergic responses orin lung or other tissue, (16) modulation, e.g., a decrease, in serum orblood of leptin levels in, e.g., obese subjects such as humans with abody mass index of about 27, 28, 29, 30, 31, 32, 33, 34 or greater, (17)increased corticotropin releasing hormone synthesis or activity in,e.g., elderly subjects such as humans at least about 60 years of age orat least about 70 years of age, (18) enhancement of memory or reductionof memory loss or disorientation in aging or dementias such asAlzheimer's Disease, (20) enhancement of the synthesis or activity ofone or more enzymes responsible for thermogenesis, e.g., liverglycerol-3-phosphate dehydrogenase or malic enzyme, in subjects such asobese or diabetic humans, (21) modulation, e.g., reduction, of thesynthesis or biological activity of the CXCR4 receptor or the CXCL12chemokine in hyperproliferation conditions such as breast cancers orprecancers, (22) modulation of the synthesis or biological activity ofone or more of holocytochrome c, cytochrome c, secondmitochondria-derived activator of caspase, Apaf-1, Bax, procaspase-9,caspase-9, procaspase-3, caspase-3, caspase-6 and caspase-7, e.g.,enhanced translocation of these molecules from mitochondria to cytosolor activation of these molecules in the cytosol in cancer precancercells, cancer cells or cells that mediate autoimmunity, (23) modulationof the synthesis or biological activity of one or more of tumor necrosisfactor-α, interleukin-1β converting enzyme, IL-6, IL-8, caspase-4 andcaspase-5, e.g., decreased activation of these molecules in injuredcells or cells subject to injury from, e.g., ischemia or infarction(e.g., vascular, cardiac or cerebral), reperfusion of hypoxic cells ortissue or an inflammation condition such as rheumatoid arthritis,ulcerative colitis, viral hepatitis, alcoholic hepatitis, or anotherinflammation condition disclosed herein, (24) decrease of the synthesis,biological activity or activation of one or more of phospholipase A2,caspase-1, caspase-3 and procaspase-3 in neurodegeneration disorders ordementias such as Alzheimer's disease, Huntington's disease, or anotherneurological condition disclosed herein. The formula 1 compounds canthus be used where one or more of these conditions or their symptoms ispresent. Methods to measure the synthesis or biological activity ofthese molecules has been described, see, e.g., U.S. Pat. Nos. 6,200,969,6,187,767, 6,174,901, 6,110,691, 6,083,735, 6,024,940, 5,919,465 and5,891,924.

The formula 1 compounds can facilitating release of myeloperoxidase fromgranulated neutrophils. The enzyme generates free hydrogen peroxide.Some of the formula 1 compounds, e.g., compounds with a halogen such asbromine or iodine at, e.g., the 16 position, can be metabolized toprovide a source of halogen. In cases where the halogen is released, thereleased halogen can react with hydrogen peroxide (H₂O₂) to generatehypohalogenous acid such as hypobromous acid (HOBr). Exemplary compoundsinclude a halogenated formula 1 compound such as16-bromoepiandrosterone. Alternatively, a halogen salt, e.g., KBr, NaBr,KI or NaI, can be administered to the subject to provide a source ofhalogen. The halogen source can be administered to a subject as acomponent in a formulation that comprises a formula 1 compound or it canbe administered separately. Hypohalogenous acid is a potentantimicrobial agent, which may be effective in reducing pathogens in thecirculatory system of subjects with a blood cell deficiency who alsohave a pathogen infection. Hypohalogenous acid that is generated in vivowould provide benefits to such subject as shown by, e.g., a reducedquantitative circulating viral or bacterial culture measurement, withoutthe toxicity that is normally associated with its direct administrationto a subject. Biological activities of white blood peroxidase enzymeshave been described, see, e.g., M. Saran et al., Free Radical Biol. Med.1999 26:482-490, W. Wu et al., J. Clin. Invest. 2000 105:1455-1463 andZ. Shen et al., Biochemistry 2000 39:5474-5482.

Delayed radiation effects. Invention embodiments include a method toprevent, treat or ameliorate a symptom or condition associated with oneor more delayed adverse effect, symptom or condition from radiationexposure in a subject in need thereof comprising administering to thesubject, or delivering to the subject's tissues, an effective amount ofa formula 1 compound. Radiation exposure may arise from a radiationtherapy where exposure is intentional, or it may arise from anaccidental exposure.

Radiation therapy (“RT”) can generate a number of early or latedelayed-onset conditions or symptoms. Delayed radiation effects areconditions or symptoms that generally arise or become detectable to thesubject or to a health care provider at least about 1 month afterexposure to radiation. Thus the conditions or symptoms may detectable atabout 2 months, about 3 months, about 4 months, about 1 year, about 20years or more after radiation exposure. For example, transient nervoussystem symptoms may develop early after RT, but progressive, permanent,often disabling nervous system damage may appear months or years later.The total radiation dose, size of the fractions, duration of RT, andvolume of tissue irradiated influence the probability of the injury andits severity. Individual patient and tissue susceptibility to delayedinjuries is variable, which factors into the selection of safe andeffective radiation doses for RT. Total radiation doses that a subjectmay receive may comprise single doses or 2, 3, 4, or more doses within arange of about 1 to about 400 Gy, e.g., about 1, 1.4, 1.6, 1.8, 2, 2.5,3, 5, 10, 20, 40, 50, 80, 100, 130, 150, 180, 200, 250, 300, 400 or moreGy. Such doses in a given course of treatment may be the same ordifferent.

In some embodiments, the total radiation dose occurs on a singleexposure that occurs in a relatively short time period, e.g., about 1-20minutes to about 12 hours. In other embodiments, the total dose isdelivered to the subject in multiple doses or over a longer time, e.g.,over about 2 days to about 12 months or more in multiple doses in, e.g.,2, 3, 4, 6, 8, 10 or more individual doses. Ameliorating a side effectmay comprise detectably slowing the progression of a symptom orcondition or detectably reducing the ultimate expected severity of asymptom or condition. The affected condition or symptom may bedetectably reduced as determined by the subject or the health careprovider. Thus, after administration of a formula 1 compound, the targetsymptom or condition may be moderately reduced, slightly reduced,essentially nonexistent or subclinical, e.g., present at a low levelthat is not deemed significant by the subject or the health careprovider. Amelioration of one or more conditions or symptoms that can besuitably quantified may be observed as a decrease of about 5% or more,e.g., at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 70%, at least about80% or at least about 90% in the relative expected or potential severityor extent of the condition or symptom.

For example, in lung pneumonitis, administration of a formula 1 compoundcan lead to detectably increased oxygen saturation in the subject'sblood by about 5% or by about 10% or more, e.g., oxygen saturation canrise from about 83% to about 88%, which would typically be detectable bythe subject and the health care provider. Such decreased severity of acondition or symptom may be objectively measured in some instances,e.g., by determining the number or activity of circulating platelets orneutrophils or by evaluation of fever, severity or frequency of diarrheaor blood oxygen saturation levels. For other symptoms or conditions,prevention may be subjectively observed by a significant or detectableimprovement in a relevant score, e.g., decreased fever or pain or adecreased need for treatment of fever, pain or inflammation.

Symptoms or conditions of radiation exposure that can be treated alsoinclude encephalopathy, myelopathy, nausea, diarrhea, acuteinflammation, chronic inflammation, edema, pain, fever, headache,depression, malaise, weakness, hair loss, skin atrophy, skin ulceration,skin lesion, keratosis, telangiectasia, infection, e.g., bacterial,viral, fungal or yeast infection, hypoplasia, atrophy, marrowhypoplasia, hemorrhage, fibrosis, e.g., lung fibrosis, pneumonitis, bonemarrow hypoplasia, hemorrhage or cytopenia, e.g., anemia, leukopenia orthrombocytopenia, edema, fibrosis or hemorrhage or the need for edema,fibrosis or hemorrhage treatment. Such symptoms or conditions may arisefrom one or more radiation-damaged tissues or cells, including lymphoidcells, bowel or intestinal epithelium or tissue, bone marrow, testicles,ovaries, brain tissue, spinal cord tissue or skin epithelium.

Exemplary symptoms or conditions associated with late effects ofradiation exposure include (1) acute or chronic radiation-inducedenteritis or diarrhea, e.g., in patients receiving pelvic radiotherapy,(2) pseudomembranous inflammation, (3) perivascular fibrosis, (4)endothelial cell damage or death, e.g., associated with vascularradiation therapy, (5) cardiac tissue inflammation or damage orpericardial disease, e.g., in pediatric or adult patients receivingradiation therapy for a leukemia, thoracic neoplasm or other malignancy,(6) pulmonary tissue inflammation or damage, (7) hematopoietic or marrowcell inflammation or damage, e.g., in wide field radiation therapy, (8)endocrine or thyroid dysfunction, e.g., in thalamic or hypothalamictumors in pediatric or other patients, (9) decreased growth or decreasedbone development or density, e.g., in pediatric patients receivingradiation therapy for a childhood leukemia or other malignancy, (10)central nervous system inflammation or damage, e.g., in pediatric oradult patients receiving radiation therapy for a leukemia (e.g., CNSacute lymphocytic leukemia) or other malignancy, (11) connective tissuedamage after radiation therapy, (12) incidence or severity of asecondary leukemia such as acute myelogenous leukemia or myelodysplasiaand (13) gastric ulceration, bleeding, small bowel obstruction orfistula formation in, e.g., patients receiving radiation therapy to thegastrointestinal tract. These symptoms or conditions are treated orameliorated using the formula 1 compounds essentially as disclosedherein.

In treating such symptoms or conditions, slowing the progression of asymptom, condition or side effect will detectably reduce the rate atwhich the condition, symptom or side effect worsens or intensifies. Insome embodiments, pronounced slowing of the rate of progression is,e.g., the time needed to progress to an expected or a measurable point,which may be increased by a period of about 1, 2, 3, 4, 5, 10, 20, 30 ormore days to a period of about 1, 2, 3, 4, 6, 8, 10, 12, 18, 24, 36, 48,72 or more months.

Radiation-associated brain damage can give rise to acute encephalopathywith symptoms such as headache, nausea, vomiting, somnolence,depression, disorientation, and worsening neurologic signs. Theencephalopathy may arise from the first, second or a subsequentradiation fraction, e.g., when high intracranial pressure has not beentreated with, e.g., corticosteroids. Late-delayed radiation damage tothe brain or nervous system can arise at about 5, 6, 7, 8, 9, or 10months to 1, 2, 3 or more years after leukemia prophylaxis in childrenor after brain tumor prophylaxis or treatment in adults. Symptoms ofteninclude pain or headache and progressive dementia without focal signsand adults typically also develop an unsteady gait. Cerebral atrophyappears on CT scans in some cases. Late-delayed damage can arise atabout 1 week, about 2 weeks about 2 months or about 1-2 years afterirradiation of extracranial tumors or high-dose irradiation ofintracranial tumors, e.g., brachytherapy or radiosurgery, although thesymptoms are generally more focal. The invention method would be usedduring the time period when such symptoms would be expected to arise,e.g., commencing at about 1-5 days or about 7-60 days after radiationexposure and ending at about 0.5, 1, 2, 3, 4, 5 or more years later.Exemplary brachytherapies and unsealed source therapies include prostate¹²⁵I seed implants in prostate conditions such as prostate cancer, ⁹⁰Ytconjugated to monoclonal antibodies or in endovascular brachialradiotherapy.

Early-delayed radiation spinal cord myelopathy follows radiation therapyto the spinal cord, neck, upper thorax or lumbar region or and it isoften characterized by Lhermitte's sign, i.e., an electric shock-likesensation radiating down the back and into the legs on neck flexion.Late-delayed radiation myelopathy can arise months or years aftertherapy for extraspinal tumors, e.g., Hodgkin's disease. Other symptomscan include progressive weakness and sensory loss, such as aBrown-Sequard type, i.e., a proprioceptive sensory loss and weakness onone side of the body and loss of temperature and pain sensation on theother side. Progression times vary, but many human patients sufferingfrom late-delayed radiation spinal cord myelopathy become paraplegic.Late-delayed radiation neuropathy may produce brachial neuropathy, e.g.,after treatment for breast or lung cancer. Radiation can also give riseto gliomas, meningiomas, or peripheral nerve sheath tumors at about 1,2, 3, 4, 5 or more years after therapy. The formula 1 compounds willgenerally be administered at about the time period when these symptomswould be expected to arise, e.g., commencing at about 1-5 days, or about7-60 days or about 6 or 12 months after radiation exposure and ending atabout 3, 4, 6 months later or about 1, 2, 3, 4, 5, 6 or more yearslater. In some embodiments, the formula 1 compound is administered tothe subject on the same day that a planned or accidental radiationexposure occurs and dosing is continued for about 1, 2, 3, 4, 8, 12 ormore weeks to about 2, 3, 4, 5, 6 or more years, or for a time asdisclosed elsewhere herein.

Early-delayed encephalopathy often arises or is detectable at about 2, 3or 4 months after radiation therapy. This encephalopathy in adults, isdistinguished from worsening or recurrent brain tumor by, e.g., computedtomography (CT) or magnetic resonance imaging (MRI). The condition inchildren can occur as a somnolence syndrome, e.g., after whole-brainirradiation for leukemia. The condition in children typically improvesspontaneously over several days to weeks. Such encephalopathies can beprevented, delayed in onset, recede more rapidly and/or be less severewhen a formula 1 compound is administered to the subject throughout theperiod when encephalopathy can arise, e.g., beginning about a week, twoweeks or a month before the expected onset of a symptom or condition andending about a week or month or two months after it would be expected toarise or to resolve.

In some embodiments, a radiation late effect is a symptom or conditionthat may arise months or years after radiation exposure, treatment withthe formula 1 compound can commence shortly, e.g., about 0.5, 1, 2, 3,4, 5, 10, 14, 21 or 28 days, after the radiation exposure or afterinitiation of a radiation treatment. In other embodiments, the inventiontreatment method can commence after radiation exposure has terminated,e.g., about 1-30 days or about 1-72 months or more after radiationexposure. In these embodiments, the treatment method can be administeredover a period of months or years, e.g., about 0.5, 1, 2, 3, 4, 5, 6, 12,18, 24, 36, 48, 72, 96 or more months. In some embodiments, dosing ofthe subject will occur for a period of about 2-12 months or for a periodof about 4-6 months. Occasionally, treatment for radiation late effectswill commence on the day of or before initiation of a planned radiationtreatment, e.g., at about 1, 2, 3, 4, 5, 7, 14, 21, 28 or more daysbefore a planned exposure to radiation of a sufficient dose to a subjectthat will potentially generate, or is likely to generate, one or moreradiation late effects, symptoms or conditions in the subject, e.g., anyradiation-associated symptom or condition disclosed herein. In any ofthese embodiments, dosing of the subject with the formula 1 compound canbe on a daily dosing basis or on an intermittent basis, e.g., using atreatment protocol essentially as described herein or in the citedreferences.

The formula 1 compounds can be used to prevent, ameliorate, slow theprogression and/or reduce the ultimate severity of marrow hypoplasia,hemorrhage, e.g., brainstem hemorrhage, cerebral hemorrhage or gastrichemorrhage or cytopenia, e.g., a blood cell count about 4-25% or morebelow the low end of a normal range for the subject, e.g., one or moreof anemia (e.g., less than about 4.0×10¹² red cells/L for adult humanfemales and less than about 4.5×10¹² red cells/L in adult human males ora hemoglobin level of less than about 12.0 g/dL in adult human femalesand less than about 13.5 g/dL in adult human males), late effectleukopenia (e.g., adult human white blood cell counts less than about3,800, 4,000 or 4,300 mm⁻³; adult human basophil counts less than about10 or 15 mm⁻³; adult human neutrophil counts less than about 1,600,1,800 or 2,000 mm⁻³; human eosinophil level less than about 100, 120 or150 mm⁻³; monocyte level less than about 260 or 300 mm⁻³) or late effectthrombocytopenia (e.g., human platelet counts less than about 15,000,18,000 or 20,000 mm⁻³).

In some embodiments, an effective amount of a formula 1 compound isadministered to a subject, or delivered to the subject's tissues,wherein the subject has received or has been exposed to a totalradiation dose of at least about 0.5 Gy to about 100 Gy or more. Theradiation dosage may comprise a single dose or two, three, four, five,six, 10 or more divided doses or subdoses. Thus, in exemplaryembodiments, the subject may have received a total radiation dose inranges of about 0.2-300 Gy, about 0.2-100 Gy, about 0.2-80 Gy 0.2-60 Gy,about 0.2-40 Gy, about 0.2-20 Gy, about 0.2-12 Gy, about 0.2-10 Gy,about 0.2-8 Gy, about 0.2-6 Gy or about 0.2-4 Gy. Subdivided doses maybe administered on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more occasions andsuch doses may be, e.g., about 0.05, 0.1, 0.3., 0.5, 0.8, 1, 2, 3, 4, 5,6 or more Gy per subdose. The subject may be exposed to radiationsubdoses over a period of about one day or over several days, e.g.,about 2, 3, 4, 5, 6, 8, 10, 20 or 25 days, or over a period of months,e.g., about 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 24, 36, 48 or moremonths. When a subject is exposed to a full dose or a subdose ofradiation, the exposure will occur over a period of about 1 minute toabout 48 hours, typically about 2-120 minutes or about 4-60 minutes.Radiation doses or subdoses may be, e.g., about 0.01, 0.05, 0.1, 0.2,0.5, 0.8, 1, 1.5, 2, 2.5, 3, 4, 5, 6 or 8 Gy per dose or subdose.

Administration of the formula 1 compound will typically commence atabout 1 day to about 6 months after a subject has received a totalradiation exposure, e.g., any dose or dose range disclosed herein.Typically, the formula 1 compound is used in the invention methodcommencing at about 2-30 days after radiation exposure or at about thetime that radiation delayed effects become apparent to the subject orthe subject's health care provider, e.g., within about 1-30 days after acondition or symptom is detected. Administration of the formula 1compound may continue for a period of about 5 days to about 60 days forconditions or symptoms that tend to resolve over a relatively short timeperiod. In other embodiments, the formula 1 compound is administered fora period of 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 24, 36, 48, 60 or moremonths for conditions or symptoms that tend to be chronic (e.g.,neurological damage or inflammation), arise over a long time period(e.g., secondary cancers or neurological damage) or to progress over arelatively long time, e.g., about 1-5 years or more (e.g., cancers orneurological damage).

In any of the radiation exposure embodiments or dosing protocolsdisclosed herein, the formula 1 compound can be administered to thesubject daily or on an intermittent basis, e.g., on about 1-5 days/weekor about 2-10 days/month. In daily dosing embodiments, the formula 1compound is administered to the subject daily for about 3 days to about5 years or longer. Exemplary daily dosing embodiments include dailyadministration of a formula 1 compound for about 14, 30, 60, 90, 120,180, 360 or more days. Daily doses may be administered in a single doseor as divided subdoses that are given, e.g., twice, three times, fourtimes or more per day. In intermittent dosing embodiments, the formula 1compound can be administered to the subject on 1, 2, 3, 4 or 5 dayswithin a one week period, followed by a period of about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 12, 16, 20, 24, 28 or 32 weeks without administration ofthe formula 1 compound, followed by administration of the formula 1compound to the subject on 1, 2, 3, 4 or 5 days within a one weekperiod. In other intermittent dosing embodiments, the formula 1 compoundis administered to the subject every other day, every two days, everythree days, every 4 days or every seven days.

For any radiation exposure situation where delayed radiation effects mayarise, e.g., a radiation exposure as disclosed herein, dailyadministration may comprise administering about 0.01 mg/kg to about 500mg/kg of the formula 1 compound to a subject per day. Exemplary dosagesare about 0.1-100 mg/kg/day and about 0.2-30 mg/kg/day. Exemplary unitdoses comprise about 1, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 200, 300or 500 mg of a formula 1 compound in a suitable formulation. Exemplaryunit dosages for humans or other subjects disclosed herein comprise aformulation that comprises about 1-1000 mg of a formula 1 compound orabout 5-400 mg or about 10-300 mg, e.g., about 5, 10, 20, 25, 30, 40,50, 60 75, 100, 150, 200, 250, 300, 400 or 500 mg. Larger unit dosages,e.g., about 10-400 mg, will generally be used with larger subjects suchas humans, while smaller subjects such as rodents or dogs will generallyutilize lower unit dosages, e.g., about 0.5-25 mg.

In some embodiments, the formula 1 compound is present in a formulationthat comprises micronized compound, e.g., compound that is milled,sieved, ground or the like. Micronized compound may be prepared usingany suitable process for micronizing, a number of which are known in theart. The micronized particles may include a percentage of particles thatare less than or equal to about 0.1-20 μm in diameter. Micronizedpreparations may comprise formula 1 compounds having an average particlesize of about 0.02, 0.05, 0.1, 0.2, 0.4, 0.5, 1, 2, 5, 10 15, 20, 30 or50 μm. Ranges of average particle sizes include formula 1 compounds ofabout 0.1-0.5 μm, about 0.1-0.4 μm, about 0.5-1 μm, about 1-20 μm orabout 2-50 μm. An alternative to micronizing a compound is to solubilizethe compound and incorporate it into liposomes of appropriate size. Themanufacture of liposomes and the insertion of active ingredients intosuch liposomes are known.

Numbered embodiments. Several aspects of the invention and relatedsubject matter includes the following numbered embodiments.

In some aspects, the invention relates to non-aqueous liquidformulations that comprise a formula 1 compound. Exemplary embodimentsare as follows.

1. A composition comprising one or more compounds of formula 1 orformula 2 and one or more nonaqueous liquid excipients, wherein thecomposition comprises less than about 3% v/v water.

2. The composition of embodiment 1 wherein the one or more formula 1compounds has the structure

wherein R⁷ and R⁹ independently are —CHR¹⁰—, —CH₂—, —CH═, —O—, —S— or—NH—, wherein R¹⁰ is —OH, —SH, C₁₋₁₀ optionally substituted alkyl, C₁₋₁₀optionally substituted alkoxy, C₁₋₁₀ optionally substituted alkenyl orC₁₋₁₀ optionally substituted alkynyl; and R⁸ is —CH₂—, —O—, —S— or —NH—,wherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are α.α.α.α (i.e., 5α, 8α, 9α, 14α), α.α.α.β, α.α.β.α,α.β.α.α, β.α.α.α, α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α,α.β.β.β, β.α.β.β, β.β.α.β, β.β.β.α or β.β.β.β, typically α.α.β.α orβ.α.β.α.

3. The composition of embodiment 2 wherein the one or more formula 1compounds has the structure

wherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α, α.α.β.β,α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β, β.β.α.β,β.β.β.α or β.β.β.β, typically α.α.β.α or β.α.β.α.

4. The composition of embodiments 1, 2 or 3 wherein one, two, three orfour formula 1 compounds are present.

5. The composition of embodiments 1, 2, 3 or 4 wherein the compositioncomprises less than about 0.3% v/v water.

6. The composition of embodiments 1, 2, 3, 4 or 5 wherein the one ormore nonaqueous liquid excipients is one, two or more of an alcohol, apolyethylene glycol, propylene glycol or benzyl benzoate.

7. The composition of any of embodiments 1-6 (embodiment 1, 2, 3, 4, 5or 6) wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7α-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7α,17β-trihydroxy-5-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one,3β,7α,-dihydroxyepiandrosterone, 3β,7β,-dihydroxyepiandrosterone,3β,-hydroxy-7-oxoepiandrosterone.

8. The composition of embodiment 7 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

9. The composition of any of embodiments 1-8 wherein the compositioncomprises two, three, four or five nonaqueous liquid excipients.

10. The composition of embodiment 9 wherein the composition comprisesthree or more nonaqueous liquid excipients.

11. The composition of any of embodiments 1-10 wherein the formula 1compound comprises about 0.0001-99% w/v of the composition.

12. The composition of any of embodiments 1-11 wherein the compositioncomprises a unit dose.

13. The composition of embodiment 12 wherein the unit dose comprisesabout 0.5-100 mg/mL of the formula 1 compound.

14. The composition of embodiment 10 wherein the composition comprisesabout 1.0-60 mg/mL of the formula 1 compound.

15. The composition of embodiment 14 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

16. The composition of embodiment 1 wherein the one or more nonaqueousliquid excipients comprise a polyethylene glycol, propylene glycol andbenzyl benzoate.

17. The composition of embodiment 16 wherein the composition comprisesless than about 0.3% v/v water.

18. The composition of embodiment 17 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

19. The composition of embodiment 18 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

20. The composition of embodiment 16 that further comprises an alcohol.

21. The composition of embodiment 20 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

22. The composition of embodiment 1 wherein the one or more nonaqueousliquid excipients comprise benzyl benzoate, a polyethylene glycol, analcohol and optionally an additional nonaqueous liquid excipient.

23. The composition of embodiment 22 wherein the composition comprisesless than about 0.3% v/v water.

24. The composition of embodiment 22 or 23 wherein the formula 1compound is 16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

25. The composition of embodiment 24 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

26. The composition of embodiment 22, 23, 24 or 25 wherein thepolyethylene glycol is polyethylene glycol 300 and/or polyethyleneglycol 200.

27. The composition of embodiment 26 wherein the alcohol is polyethyleneglycol is polyethylene glycol 300.

28. The composition of embodiments 22 or 23 that comprises about 2.5-25%v/v ethanol, about 1-10% v/v benzyl benzoate, about 10-35% v/vpolyethylene glycol 300, about 40-65% v/v propylene glycol and about2-60 mg/mL 16α-bromo-3β-hydroxy-5α-androstan-17-one.

28A. The composition of embodiments 22, 23, 24, 25 or 26 that comprisesabout 0.1-10% v/v benzyl benzoate, about 0.1-10% v/v benzyl alcohol,about 1-95% v/v polyethylene glycol 200, about 1-95% v/v propyleneglycol and about 2-60 mg/mL 16α-bromo-3β-hydroxy-5α-androstan-17-one.The embodiment 28A composition may comprise about 2% v/v benzylbenzoate, about 2% v/v benzyl alcohol, about 40% v/v polyethylene glycol200, about 51% v/v propylene glycol (qs) and about 50 mg/mL16α-bromo-3β-hydroxy-5α-androstan-17-one.

29. The composition of embodiment 28 that comprises about 12.5% v/vethanol, about 5% v/v benzyl benzoate, about 25% v/v polyethylene glycol300, about 57.5% v/v propylene glycol and about 50 mg/mL16α-bromo-3β-hydroxy-5α-androstan-17-one.

30. The composition of any of embodiments 1-29 that further comprises alocal anesthetic.

31. The composition of embodiment 30 wherein the local anesthetic isprocaine, benzocaine or lidocaine.

32. The composition of any of embodiments 1-31 wherein the compositioncomprises a solvate, a suspension, a colloid, a gel or a combination ofany of the foregoing.

33. A product produced by the process of contacting a compositioncomprising one or more compounds of formula 1 and a first nonaqueousliquid excipient with a second nonaqueous liquid excipient wherein theproduct comprises less than about 3% water and the salts, analogs,configurational isomers and tautomers thereof.

34. The product of embodiment 33 wherein the product comprises less thanabout 0.3% water.

35. The product of embodiments 33 or 34 wherein the first nonaqueousliquid excipient is a polyethylene glycol (e.g., PEG300 or PEG 200) orpropylene glycol.

36. The product of embodiments 33, 34 or 35 wherein the secondnonaqueous liquid excipient is a polyethylene glycol (e.g., PEG300 orPEG 200) or propylene glycol.

38. A product produced by the process of contacting a compositioncomprising one or more compounds of formula 1 and two nonaqueous liquidexcipients with a third nonaqueous liquid excipient wherein the productcomprises less than about 3% water and the salts, analogs,configurational isomers and tautomers thereof.

39. The product of embodiment 38 wherein the product comprises less thanabout 0.3% water.

40. The product of embodiments 38 or 39 wherein the two nonaqueousliquid excipients are selected from a polyethylene glycol (e.g., PEG300or PEG 200), propylene glycol, benzyl benzoate and an alcohol (e.g.,ethanol).

41. The product of embodiments 38, 39 or 40 wherein the third nonaqueousliquid excipient is a polyethylene glycol (e.g., PEG300 or PEG 200),propylene glycol, benzyl benzoate or an alcohol (e.g., ethanol).

42. A product produced by the process of contacting a compositioncomprising one or more compounds of formula 1 and three nonaqueousliquid excipients with a fourth nonaqueous liquid excipient wherein theproduct comprises less than about 3% water and the salts, analogs,configurational isomers and tautomers thereof.

43. The product of embodiment 42 wherein the product comprises less thanabout 0.3% water.

44. The product of embodiments 42 or 43 wherein the three nonaqueousliquid excipients are selected from a polyethylene glycol (e.g., PEG300or PEG 200), propylene glycol, benzyl benzoate and an alcohol (e.g.,ethanol).

45. The product of embodiments 42, 43 or 44 wherein the fourthnonaqueous liquid excipient is a polyethylene glycol (e.g., PEG300 orPEG 200), propylene glycol, benzyl benzoate or an alcohol (e.g.,ethanol).

46. The product of any of embodiments 33-45 wherein the product has beenstored at reduced temperature (about 4° C. to about 8° C.) or at ambienttemperature for about 30 minutes to about 2 years.

47. The product of any of embodiments 33-46 wherein the one or morecompounds of formula 1 comprise 1, 2, 3 or 4 formula 1 compounds.

48. The product of any of embodiments 33-46 wherein the one or morecompounds of formula 1 comprises one formula 1 compound.

49. The product of any of embodiments 33-48 wherein the one or moreformula 1 compound is selected from16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,176-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one and16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

50. The product of embodiment 49 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

51. The product of embodiment 49 that comprises about 2.5-25% v/vethanol, about 1-10% v/v benzyl benzoate, about 10-35% v/v polyethyleneglycol 300, about 40-65% v/v propylene glycol and about 2-60 mg/mL16α-bromo-3β-hydroxy-5α-androstan-17-one.

52. The product of embodiment 51 that comprises about 12.5% v/v ethanol,about 5% v/v benzyl benzoate, about 25% v/v polyethylene glycol 300,about 57.5% v/v propylene glycol and about 50 mg/mL16α-bromo-3β-hydroxy-5α-androstan-17-one.

53. The product of any of embodiments 33-52 that further comprises alocal anesthetic.

54. The composition of 52 wherein the local anesthetic is procaine,benzocaine or lidocaine.

55. A product produced by the process of contacting a compositioncomprising a compound of formula 1 with a nonaqueous liquid excipientwherein the product comprises less than about 3% v/v water and thesalts, analogs, configurational isomers and tautomers thereof.

56. The product of embodiment 55 wherein the product comprises less thanabout 0.3% v/v water.

57. The product of embodiment 53 wherein the product has been stored atreduced temperature (about 0° C. to about 8° C.) or at ambienttemperature for about 1 hour to about 2 years.

58. The product of embodiment 53 wherein the first nonaqueous liquidexcipient is a polyethylene glycol, an alcohol, propylene glycol orbenzyl benzoate.

59. The product of any of embodiments 33-58 wherein the formula 1compound comprises about 0.01% to about 99% w/v of the product.

60. The product of any of embodiments 33-59 wherein the product is aunit dose.

61. The unit dose of embodiment 60 comprising a solution containingabout 0.5-70 mg/mL of the one or more formula 1 compound.

62. The product of any of embodiments 55-61 wherein the one or moreformula 1 compound is selected from16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one and16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

63. The product of embodiment 62 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

64. The product of any of embodiments 33-61 wherein the one or moreformula 1 compound is selected from the compounds or one or more of thespecies of compounds within the genera named in compound groups 1through 21-10-6.

65. A method comprising administering the composition or product of anyof embodiments 1-64 to a subject suffering from a pathogen infection ora malignancy or an immune suppression or disregulation condition, e.g.,a suppressed Th1 immune response or an unwanted Th2 immune response.

66. The method of embodiment 65 wherein the pathogen infection is a DNAvirus infection or an RNA virus infection.

67. The method of embodiment 66 wherein the RNA virus infection is aretrovirus infection or a hepatitis virus infection.

68. The method of embodiment 67 wherein the retrovirus infection orhepatitis virus infection is an HIV, FIV, SIV, SHIV or hepatitis C virusinfection.

69. The method of embodiment 65 wherein the pathogen infection is anintracellular parasite infection.

70. The method of embodiment 69 wherein the intracellular parasiteinfection is a malaria infection.

71. The method of embodiment 65 wherein the formula 1 compound has thestructure

wherein one, two or three of R⁷, R⁸ and R⁹ are —CH₂— or —CH═ and whereinthe configuration of hydrogen atoms at the 5 (if present), 8, 9 and 14positions respectively are α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α,α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β,β.β.α.β, β.β.β.α or β.β.β.β, typically α.α.β.α or β.α.β.α.

72. The method of embodiment 71 wherein the formula 1 compound has thestructure

73. The method of embodiment 72 wherein R¹, R² and R⁴ independently are—OH, aC2-C20 ester or C1-C20 alkoxy, R³ is —H and two or three of R⁷, R⁸and R⁹ are —CH₂—.

74. The method of embodiment 72 or 73 wherein the formula 1 compound hasthe structure

75. The method of any of embodiments 71-74 wherein the configuration ofhydrogen atoms at the 5 (if present), 8, 9 and 14 positions respectivelyare α.α.β.α or β.α.β.α.

In other embodiments, the formula 1 compounds include new compounds,some of which are described in the following numbered embodiments.

1A. A compound of formula 1 having the structure

wherein R⁷, R⁸ and R⁹ are independently selected and wherein one, two orthree of R⁷, R⁸ and R⁹ are not —CH₂— or —CH═ and wherein hydrogen atomsat the 5 (if present), 8, 9 and 14 positions respectively are in theα.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α, α.α.β.β, α.β.α.β, β.α.α.β,β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β, β.β.α.β, β.β.β.α orβ.β.β.β, typically α.α.β.α or β.α.β.α.

2A. The compound of embodiment 1A wherein R⁸ is —CH₂—, —O—, —S— or —NH—.

3A. The compound of embodiment 1A or 2A wherein R⁷ is —CH₂—CHR¹⁰—,—O—CHR¹⁰— or —O—C(O)—.

4A. The compound of embodiment 1A, 2A or 3A wherein R⁸ or R⁹ is absent.

5A. The compound of embodiment 1A or 2A wherein R⁷ and R⁹ independentlyare —CHR¹⁰ —, —CH₂—, —CH═, —O—, —S— or —NH—, wherein R¹⁰ is —OH, —SH, aC₁₋₃₀ organic moiety, a C₁₋₃₀ ester, C₁₋₁₀ optionally substituted alkyl,C₁₋₁₀ optionally substituted alkoxy, C₁₋₁₀ optionally substitutedalkenyl or C₁₋₁₀ optionally substituted alkynyl.

6A. The compound of embodiment 1A, 2A, 3A, 4A or 5A wherein the formula1 compound has the structure

wherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are in the α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α,α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β,β.β.α.β, β.β.β.α or β.β.β.β configurations, typically α.α.β.α orβ.α.β.α.

7A. The compound of embodiment 6A wherein R⁴ is —OH, ═O, —SH, a C₁₋₃₀ester or C₁₋₃₀ alkoxy, wherein the ester or alkoxy moiety is optionallysubstituted with one, two or more independently selected substituents,which are optionally selected from —F, —Cl, —Br, —I, —O—, ═O, —S—, —NH—,—OR^(PR), —SR^(PR) or —NHR^(PR).

8A. The compound of embodiment 6A or 7A wherein R¹ is —OH, ═O, —SH, aC₁₋₃₀ ester or C₁₋₃₀ alkoxy, wherein the ester or alkoxy moiety isoptionally substituted with one, two or more independently selectedsubstituents, which are optionally selected from —F, —Cl, —Br, —I, —O—,═O, —S—, —NH—, —OR^(PR), —SR^(PR) or —NHR^(PR).

9A. The compound of embodiment 1A, 2A or 3A wherein the formula 1compound has the structure

wherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α, α.α.β.β,α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β, β.β.α.β,β.β.β.α or β.β.β.β, typically α.α.β.α or β.α.β.α.

10A. The compound of embodiment 9A wherein R⁴ is —OH, ═O, —SH, a C₁₋₃₀ester or C₁₋₃₀ alkoxy, wherein the ester or alkoxy moiety is optionallysubstituted with one, two or more independently selected substituents,which are optionally selected from —F, —Cl, —Br, —I, —O—, ═O, —S—, —NH—,—OR^(PR), —SR^(PR) or —NHR^(PR).

11A. The compound of embodiment 9A or 10A wherein R¹ is —OH, ═O, —SH, aC₁₋₃₀ ester or C₁₋₃₀ alkoxy, wherein the ester or alkoxy moiety isoptionally substituted with one, two or more independently selectedsubstituents, which are optionally selected from —F, —Cl, —Br, —I, —O—,═O, —S—, —NH—, —OR^(PR), —SR^(PR) or —NHR^(PR).

12A. A composition comprising a compound of any of embodiments 1A-11Aand an excipient suitable for human pharmaceutical use or for veterinaryuse, e.g., an excipient disclosed herein or in the cited references.

13A. A product produced by the process of contacting a compound of anyof embodiments 1A-11A and an excipient suitable for human pharmaceuticaluse or for veterinary use, e.g., an excipient disclosed herein or in thecited references.

14A. The use of a compound, composition or product of any of embodiments1A-13A to prepare a medicament for use to prevent or to treat, or toameliorate one or more symptoms associated, with an infection, animmunosuppression condition, a malignancy, a pre-malignant condition orto modulate a mammal's immune response, such as enhancing a Th1 responseor decreasing a Th2 response, e.g., an infection, malignancy or immunedysregulation as described herein or in the cited references.

15A. The use of embodiment 1 4A, wherein the infection is a viralinfection (e.g., HIV, HCV, a Herpesvirus, a togavirus, a human papillomavirus infection or other virus described herein or in the citedreferences), a bacterial infection (e.g., Borrelia sp., Legionella sp.or other bacterium described herein or in the cited references), afungal or a yeast infection (e.g., Candida sp., Aspergillus sp. or otheryeast described herein or in the cited references) or a parasiteinfection (e.g., a malaria parasite, a gastrointestinal nematode, ahelminth, Leishmania sp., Cryptosporidium sp., Toxoplasma gondii,Pneumocystis Schistosoma sp. (e.g., S. mansoni, S. japonicum, S.haematobium), Strongyloides stercoralis or other parasite describedherein or in the cited references).

16A. The compound, composition, product or use of any of embodiments1A-15A, wherein the formula 1 compound is a compound named in any ofcompound groups 1 through54-53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6,or the formula 1 compound is a species in any genus described in any ofcompound groups 1 through54-53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6.

In other aspects, the invention provides dosing methods suitable totreat the conditions described herein. The following embodimentsdescribe some of these methods.

1B. A method comprising intermittently administering one or morecompounds of formula 1 (or a composition comprising a formula 1compound) to a subject or delivering to the subject's tissues a formula1 compound(s) (or a composition comprising a formula 1 compound), e.g.,any formula 1 compound named or described herein, or a metabolicprecursor or a biologically active metabolite thereof, including thecompounds described in embodiments 1-64 and 1A-11A above.

2B. The method of embodiment 1B wherein the subject has an infection, ahyperproliferation disorder, a hypoproliferation condition, animmunosuppression condition, an unwanted immune response or wherein thesubject has recently experienced or will shortly experience trauma,surgery or a therapeutic treatment wherein the therapeutic treatment isone other than the method of embodiment 1B.

3B. The method of embodiment 2B wherein the immunosuppression conditionor the unwanted immune response is associated with a viral infection, anintracellular bacterial infection, an extracellular bacterial infection,a fungal infection, a yeast infection, an extracellular parasiteinfection, an intracellular parasite infection, a protozoan parasite, amulticellular parasite, an autoimmune disease, a cancer, a precancer, achemotherapy, a radiation therapy, an immunosuppressive therapy, ananti-infective agent therapy, a wound, a burn, the presence of animmunosuppressive molecule, gastrointestinal irritation or aninflammation condition optionally selected from or associated withirritable bowel disease, Crohn's disease or chronic diarrhea, or anycombination of the foregoing.

4B. The method of embodiment 3B wherein the subject's immunosuppressioncondition is ameliorated or the unwanted immune response (e.g., a Th2response) is reduced or wherein the subject's Th1 immune response isenhanced.

5B. The method of embodiment 3B wherein the subject's innate immunity,specific immunity or both is enhanced.

6B. The method of embodiment 5B wherein the subject's innate immunity isenhanced.

7B. The method of embodiment 6B wherein the subject's specific immunityis enhanced, e.g., wherein the subject's Th2 immune response is reducedor wherein the subject's Th1 immune response is enhanced.

8B. The method of embodiment 2B wherein the one or more compounds offormula 1 is or are administered according to the a dosing regimencomprising the steps, (a) administering the one or more compounds offormula 1 to the subject at least once per day for at least 2 days; (b)not administering the one or more formula 1 compounds to the subject forat least 1 day; (c) administering the one or more formula 1 compounds tothe subject at least once per day for at least 2 days; and (d)optionally repeating steps (a), (b) and (c) at least once or variationsof steps (a), (b) and (c) at least once.

9B. The method of embodiment 8B wherein step (c) comprises the samedosing regimen as step (a).

10B. The method of embodiment 9B wherein step (a) of the dosing regimencomprises administering the one or more formula 1 compounds once perday, twice per day, three times per day or four times per day.

11B. The method of embodiment 10B wherein step (a) of the dosing regimencomprises administering the one or more formula 1 compounds once per dayor twice per day.

12B. The method of embodiment 10B wherein step (a) comprisesadministering the one or more formula 1 compounds for about 3 to about24 days.

13B. The method of embodiment 12B wherein step (a) comprisesadministering the one or more formula 1 compounds for about 4 to about12 days.

14B. The method of embodiment 13B wherein step (a) comprisesadministering the one or more formula 1 compounds for about 4 to about 8days.

15B. The method of embodiment 14B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 3 to about120 days.

16B. The method of embodiment 15B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 4 to about60 days.

17B. The method of embodiment 16B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 5 to about30 days.

18B. The method of embodiment 16B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 8 to about60 days. 19B.

The method of embodiment 15B wherein steps (a), (b), and (c) arerepeated at least about 4 times.

20B. The method of embodiment 15B wherein steps (a), (b), and (c) arerepeated about 5 times to about 25 times.

21B. The method of embodiment 15B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 2 months.

22B. The method of embodiment 15B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 12 months.

23B. The method of embodiment 8B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 3 to about120 days.

24B. The method of embodiment 23B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 4 to about60 days.

25B. The method of embodiment 24B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 5 to about30 days.

26B. The method of embodiment 23B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 8 to about60 days.

27B. The method of embodiment 8B wherein step (d) comprises repeatingsteps (a), (b), and (c) at least once.

28B. The method of embodiment 27B wherein step (d) comprises repeatingsteps (a), (b), and (c) about 3 times to about 25 times.

29B. The method of embodiment 1B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 2 months.

30B. The method of embodiment 29B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 12 months.

31B. The method of any of embodiments 8B-30B wherein theimmunosuppression condition or the unwanted immune response isassociated with a viral infection, an intracellular bacterial infection,an extracellular bacterial infection, a fungal infection, a yeastinfection, an extracellular parasite infection, an intracellularparasite infection, a protozoan parasite, a multicellular parasite, anautoimmune disease, a cancer, a precancer, a chemotherapy, a radiationtherapy, an immunosuppressive therapy, an anti-infective agent therapy,a wound, a burn, the presence of an immunosuppressive molecule,gastrointestinal irritation or an inflammation condition optionallyselected from or associated with irritable bowel disease, Crohn'sdisease or chronic diarrhea, or any combination of the foregoing.

32B. The method of embodiment 31B wherein the subject'simmunosuppression condition is ameliorated or the unwanted immuneresponse is reduced.

33B. The method of embodiment 32B wherein the subject's innate immunity,specific immunity or both is enhanced.

34B. The method of embodiment 33B wherein the subject's innate immunityis enhanced.

35B. The method of embodiment 34B wherein the subject's specificimmunity is enhanced.

36B. The method of embodiment 8B wherein step (c) comprises the ashorter dosing regimen than step (a).

37B. The method of embodiment 36B wherein step (a) comprisesadministering the formula 1 compound for 7 to about 24 days.

38B. The method of embodiment 37B wherein step (c) comprisesadministering the formula 1 compound for 4 to about 12 days.

39B. The method of embodiment 38B wherein step (b) comprises notadministering the formula 1 compound for about 3 to about 120 days.

40B. The method of embodiment 39B wherein step (b) comprises notadministering the formula 1 compound for about 4 to about 60 days.

41B. The method of embodiment 40B wherein step (b) comprises notadministering the formula 1 compound for about 5 to about 30 days.

42B. The method of embodiment 36B wherein step (d) comprises repeatingsteps (a), (b), and (c) at least once.

43B. The method of embodiment 42B wherein step (d) comprises repeatingsteps (a), (b), and (c) about 3 times to about 25 times.

44B. The method of embodiment 36B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 2 months.

45B. The method of embodiment 44B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 12 months.

46B. The method of any of embodiments 36B-45B wherein theimmunosuppression condition or the unwanted immune response isassociated with a viral infection, an intracellular bacterial infection,an extracellular bacterial infection, a fungal infection, a yeastinfection, an extracellular parasite infection, an intracellularparasite infection, a protozoan parasite, a multicellular parasite, anautoimmune disease, a cancer, a precancer, a chemotherapy, a radiationtherapy, an immunosuppressive therapy, an anti-infective agent therapy,a wound, a burn, the presence of an immunosuppressive molecule,gastrointestinal irritation or an inflammation condition optionallyselected from or associated with irritable bowel, Crohn's disease,chronic diarrhea, or any combination of the foregoing.

47B. The method of embodiment 46B wherein the subject'simmunosuppression condition is ameliorated or the unwanted immuneresponse is reduced.

48B. The method of embodiment 47B wherein the subject's innate immunity,specific immunity or both is enhanced.

49B. The method of embodiment 48B wherein the subject's innate immunityis enhanced.

50B. The method of embodiment 48B wherein the subject's specificimmunity is enhanced.

51B. The method of embodiment 8B wherein step (c) comprises a longerdosing period than step (a).

52B. The method of embodiment 51B wherein step (a) comprisesadministering the formula 1 compound for 7 to about 24 days.

53B. The method of embodiment 52B wherein step (c) comprisesadministering the formula 1 compound for 4 to about 12 days.

54B. The method of embodiment 53B wherein step (b) comprises notadministering the formula 1 compound for about 3 to about 120 days.

55B. The method of embodiment 54B wherein step (b) comprises notadministering the formula 1 compound for about 4 to about 60 days.

56B. The method of embodiment 55B wherein step (b) comprises notadministering the formula 1 compound for about 5 to about 30 days.

57B. The method of embodiment 51B wherein step (d) comprises repeatingsteps (a), (b), and (c) at least once.

58B. The method of embodiment 57B wherein step (d) comprises repeatingsteps (a), (b), and (c) about 3 times to about 25 times.

59B. The method of embodiment 51B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 2 months.

60B. The method of embodiment 59B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 12 months.

61B. The method of any of embodiments 51B-60B wherein theimmunosuppression condition or the unwanted immune response isassociated with a viral infection, an intracellular bacterial infection,an extracellular bacterial infection, a fungal infection, a yeastinfection, an extracellular parasite infection, an intracellularparasite infection, a protozoan parasite, a multicellular parasite, anautoimmune disease, a cancer, a precancer, a chemotherapy, a radiationtherapy, an immunosuppressive therapy, an anti-infective agent therapy,a wound, a burn, the presence of an immunosuppressive molecule,gastrointestinal irritation or an inflammation condition optionallyselected from or associated with irritable bowel disease, Crohn'sdisease or chronic diarrhea, or any combination of the foregoing.

62B. The method of embodiment 61B wherein the subject'simmunosuppression condition is ameliorated or the unwanted immuneresponse is reduced.

63B. The method of embodiment 62B wherein the subject's innate immunity,specific immunity or both is enhanced or wherein the subject's Th1immune response is enhanced or the subject's Th2 immune response isdecreased.

64B. The method of embodiment 8B wherein the variations of steps (a),(b) and (c) comprise conducting a first dosing regimen of steps (a), (b)and (c) once, twice or three times, followed by one or more seconddosing regimens of steps (a′), (b′) and (c′) wherein one or more of the(a′), (b′) and (c′) steps in the second dosing regimen is longer thanthe corresponding step in the first dosing regimen.

65B. The method of embodiment 8B wherein the variations of steps (a),(b) and (c) comprise conducting a first dosing regimen of steps (a), (b)and (c) once, twice or three times, followed by one or more seconddosing regimens of steps (a′), (b′) and (c′) wherein one or more of the(a′), (b′) and (c′) steps in the second dosing regimen is shorter thanthe corresponding step in the first dosing regimen.

66B. The method of any of embodiments 1B-67B wherein the one or moreformula 1 compounds is or are administered orally, intramuscularly,intravenously, subcutaneously, topically, vaginally, rectally,intracranially, intrathecally, intradermally, as an aerosol or by abuccal route.

67B. The method of embodiment 66B wherein the one or more formula 1compounds is or are present in a solid formulation predominantly as asolid or the one or more formula 1 compounds is or are present in aliquid formulation predominantly as a solvate, colloid or a suspensionor the one or more formula 1 compounds is or are present in a gel, creamor paste.

68B. The method of any of embodiments 2B-67B wherein the subject's viralinfection, intracellular bacterial infection, extracellular bacterialinfection, fungal infection, yeast infection, extracellular parasiteinfection, intracellular parasite infection, protozoan parasite,multicellular parasite, autoimmune disease, cancer, precancer,chemotherapy, radiation therapy, immunosuppressive therapy,anti-infective agent therapy, a wound, a burn, or the presence of animmunosuppressive molecule, gastrointestinal irritation or aninflammation condition optionally selected from or associated withirritable bowel disease, Crohn's disease or chronic diarrhea, or anycombination of the foregoing is (a) a DNA virus infection or an RNAvirus infection (HSV, CMV, HBV, HCV, HIV, SHIV, SIV); (b) a Mycoplasmainfection, a Listeria infection or a Mycobacterium infection; (c)extracellular bacteria infection; (d) fungal infection; (e) a yeastinfection (Candida, Cryptococcus); (d) protozoa (malaria, Leishmania,Cryptosporidium, Toxoplasmosis, Pneumocystis); (e) a multicellularparasite; (f) autoimmune diseases (SLE, RA, diabetes); (g) cancers(solid cancers selected from, e.g., ovarian, breast, prostate, glioma;disseminated cancers selected from lymphoma, leukemia, colon cancer,sarcoma); (h) precancers; (i) chemotherapies (adriamycin, cisplatin,mitomycin C); (j) radiation therapies; (k) immunosuppressive therapies;(I) anti-infective agent therapies; (m) wounds (surgical or otherwise);(n) 1^(st) degree, 2^(nd) degree or 3^(rd) degree burns; (o)immunosuppressive molecules; (p) gastrointestinal irritation or aninflammation condition optionally selected from or associated withirritable bowel, Crohn's disease, chronic diarrhea; or (q) anycombination of (a) through (p).

69B. The method of embodiment 68B wherein the RNA virus infection is aretroviral infection or a hepatitis virus infection.

70B. The method of embodiment 68B or 69B wherein the one or more formula1 compounds is one formula 1 compound.

71B. The method of embodiment 70B wherein the one or more formula 1compounds is or are in a composition that comprises, (a) one or morenonaqueous liquid excipients, wherein the composition comprises lessthan about 3% v/v water; (b) a solid that comprises a pharmaceuticallyacceptable excipient; or (c) one or more liquid excipients, wherein thecomposition comprises more than about 3% v/v water.

72B. The method of embodiment 68B or 71B wherein the formula 1 compoundis 16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

73B. The method of embodiment 72B wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

74B. The method of embodiments 1B-73B wherein the formula 1 compoundexcludes one or more of any formula 1 compounds.

75B. A method to treat involuntary weight loss, oral lesions, skinlesions, opportunistic infections, diarrhea or fatigue in an subjectcomprising intermittently administering one or more compounds of formula1 to the subject (e.g., involuntary weight loss from viral infection,gastrointestinal infection, chemotherapy, anorexia, gastrointestinalirritation or an inflammation condition optionally selected from orassociated with irritable bowel, Crohn's disease, chronic diarrhea).

76B. The method of embodiment 75B wherein the subject has animmunosuppression condition.

77B. The method of embodiment 76B wherein the subject is a human.

78B. The method of embodiment 77B wherein the subject is a human 1 dayto 18 years old (e.g., 1 month to 6 years old).

79B. The method of any of embodiments 75B-78B wherein the subject'sspecific immunity remains impaired compared to a typical comparablecontrol subject who does not have the subject's pathological condition.

80B. The method of embodiment 79B wherein the subject's CD4 cell countdoes not increase significantly during one or more courses of dosing(e.g., dosing for 1 week to about 2 weeks or more).

81B. The method of clam 80B wherein the subject's CD4 cell count isabout 20 to about 100 CD4⁺ cells/mm³ or about 20 to about 75 CD4⁺cells/mm³.

82B. The method of any of embodiments 1B-81B wherein the subject has apathogen(s) infection or a malignancy and the pathogen(s) or malignancydoes not become resistant to the formula 1 compound over a time normallyassociated with the development of measurable resistance in at leastabout 50% of subjects who are treated with a therapeutic treatment(s)other than a formula 1 compound(s).

83B. The method of embodiment 82B wherein the pathogen infection is anHIV, SIV, SHIV or HCV infection.

84B. The method of embodiments 82B or 83B wherein the formula 1 compoundis one or more of 16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one or a physiologicallyacceptable ester, carbonate, carbamate, amino acid conjugate or peptideconjugate thereof.

85B. The method of embodiment 84B wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one or a physiologically acceptableester, carbonate, carbamate, amino acid conjugate or peptide conjugatethereof.

86B. The method of any of embodiments 1B-85B or 87B-90B wherein theformula 1 compound is a compound named in any of compound groups 1through54-53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10⁻⁸-6,or the formula 1 compound is a species in any genus described in any ofcompound groups 1 through54-53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10⁻⁸-6.

87B. A method to treat, ameliorate, prevent or slow the progression ofan unwanted condition or symptom associated with the presence of anatural or synthetic glucocorticosteroid (‘GCS’) in a subject comprisingadministering to the subject, or delivering to the subject's tissues, aneffective amount of a formula 1 compound, whereby the condition orsymptom is detectably treated, ameliorated, prevented or its progressionis detectably slowed.

88B. The method of embodiment 87B wherein the unwanted condition orsymptom associated with the presence of the natural or synthetic GCS isan immune suppression disease, condition or symptom.

89B. The method of embodiment 88B wherein the immune suppressiondisease, condition or symptom is associated with a pathogen infection, acancer or precancer, aging, trauma (stress, burn, surgery, accidentaltissue injury) or inflammation.

90B. The method of embodiment 89B wherein the chemotherapy comprisestreatment of the subject with a GCS.

In other embodiments, the invention provides methods to modulate immunecells or immune responses in a subject. The following numberedembodiments describe some of these methods.

1C. A method to modulate a subject's innate immunity or to enhance asubject's Th1 immune response or to reduce a subject's Th2 immuneresponses comprising administering to the subject a compound(s) offormula 1, or a metabolic precursor or a biologically active metabolitethereof, including any formula 1 compound that is described or disclosedherein, including the compounds described in embodiments 1-64 and 1A-11Aabove.

2C. The method of embodiment 1C wherein the subject's innate immunity isenhanced.

3C. The method of embodiment 1C or 2C wherein the subject suffers froman innate immunity suppression condition, a suppressed Th1 immuneresponse or an unwanted Th2 immune response.

4C. The method of embodiment 3C wherein the innate immunity suppressioncondition, the suppressed Th1 immune response or the unwanted Th2response is associated with a viral infection, an intracellularbacterial infection, an extracellular bacterial infection, a fungalinfection, a yeast infection, an extracellular parasite infection, anintracellular parasite infection, a protozoan parasite, a multicellularparasite, an autoimmune disease, a cancer, a precancer, a chemotherapy,a radiation therapy, an immunosuppressive therapy, an anti-infectiveagent therapy, a wound, a burn, the presence of an immunosuppressivemolecule or any combination of the foregoing.

5C. The method of any of embodiments 1C-3C wherein the subject's Th1immune response is enhanced.

6C. The method of embodiment 1C wherein the subject's Th2 immuneresponse is reduced.

7C. The method of embodiment 6C wherein the subject has a conditioncomprising an unwanted immune response (e.g., autoimmune disease, SLE,diabetes).

9C. The method of embodiment 6C or 7C wherein the subject is avertebrate, a mammal, a primate or a human.

10C. The method of embodiment 9 wherein the vertebrate's, the mammal'sthe primate's or the human's specific immunity modulation is (i) anenhanced CTL or Th1 response to a virus infection or to a malignant cellin vitro or in vivo, (ii) enhanced antigen presentation or biologicalactivity by dendritic cells or dendritic cell precursors, or (iii)enhanced killing of virus-infected or of malignant cells.

11C. The method of 10C wherein the vertebrate is a human, the virusinfection is an HIV infection and the CTL or Th1 response comprises anenhanced response to one or more of the HIV's gag protein or to theHIV's gp120.

12C. The method of embodiment 1C, 4C, 10C or 11C wherein the subject'sTh1 cells, tumor-infiltrating lymphocytes (TIL cells), NK cells,peripheral blood lymphocytes, phagocytes, monocytes, macrophage,neutrophils, eosinophils, dendritic cells or fibrocytes are activated asmeasured by, e.g., enhanced ³H-thymidine uptake compared to untreatedcontrols or by an increase in the number of the cell type in circulationor demonstrable movement of the cell type from one tissue or compartment(e.g., skin) to another tissue or compartment (e.g., blood, lymph node,spleen or thymus).

13C. The method of embodiment 1C, 4C, 10C, 11C or 12C, wherein theformula 1 compound(s) modulates transcription of one or more genes inthe subject's NK cells, phagocytes, monocytes, macrophages, neutrophils,eosinophils, dendritic cells or fibrocytes are detectably activated(e.g., as measured by increased protein kinase C activity or bymodulation of a biological activity of a steroid receptor or an orphannuclear hormone receptor).

14C. The method of embodiment 1C wherein the formula 1 compound(s)enhances lysosome movement in one or more of the subject's NK cells,phagocytes, monocytes, macrophages, neutrophils, eosinophils, dendriticcells or fibrocytes.

15C. The method of embodiment 1C wherein the formula 1 compound(s)enhances protein kinase C activity in one or more of the subject's NKcells, phagocytes, monocytes, macrophages, neutrophils, eosinophils,dendritic cells or fibrocytes (e.g., PKCα, PKCβ, PKCγ and PKCζ).

16C. A composition comprising a partially purified or a purified complexcomprising a formula 1 compound and a steroid receptor, a serumsteroid-binding protein (e.g., human serum albumin, α1-acidglycoprotein, sex hormone-binding globulin, testosterone-bindingglobulin, corticosteroid-binding globulin, androgen binding protein(rat)) or a binding partner (e.g., complexing agent, liposome,antibody).

17C. A product produced by the process of contacting the partiallypurified or the purified composition of embodiment 16C with one or moresterile containers, one or more syringes, one or more pharmaceuticallyacceptable excipients (e.g., excipient as defined in draft spec aboveand including sugars, lactose, sucrose, fillers, lubricants, binders, orany excipient named in any reference cited herein), one or more cells,one or more tissues, plasma or blood.

18C. The method of any of embodiments 1C-17C wherein the subject has aninfection, a hyperproliferation disorder, a hypoproliferation condition,an immunosuppression condition, an unwanted immune response or whereinthe subject has recently experienced or will shortly experience trauma,surgery or a therapeutic treatment wherein the therapeutic treatment isone other than the method of embodiment 1C.

19C. The method of embodiment 18C wherein the immunosuppressioncondition or the unwanted immune response is associated with a viralinfection, an intracellular bacterial infection, an extracellularbacterial infection, a fungal infection, a yeast infection, anextracellular parasite infection, an intracellular parasite infection, aprotozoan parasite, a multicellular parasite, an autoimmune disease, acancer, a precancer, a chemotherapy, a radiation therapy, animmunosuppressive therapy, an anti-infective agent therapy, a wound, aburn, the presence of an immunosuppressive molecule, gastrointestinalirritation or an inflammation condition optionally selected from orassociated with irritable bowel disease, Crohn's disease or chronicdiarrhea, or any combination of the foregoing.

20C. The method of embodiment 19C wherein the subject'simmunosuppression condition is ameliorated or the unwanted immuneresponse is reduced.

21C. The method of embodiment 19C wherein the subject'simmunosuppression condition is associated with a viral infection.

22C. The method of embodiment 21C wherein the viral infection comprisesa DNA virus or an RNA virus infection.

23C. The method of embodiment 22C wherein the RNA virus infectioncomprises a retroviral infection or a hepatitis virus infection.

24C. The method of any of embodiments 18C-23C wherein the subjectsuffers from one or more of chronic diarrhea, involuntary weight loss(usually at least about 5% or more), cachexia (usually at least about 5%or more), muscle wasting, one or more oral lesions (usually at leastabout 1 cm²), one or more genital lesions (usually at least about 1cm²), skin lesions (usually at least about 1 cm²) or an opportunisticinfection associated with AIDS.

25C. A method (e.g., to determine a biological activity of a formula 1compound or to modulate transcription of a gene in a cell or cell-freetranscription system) comprising: (a) contacting the formula 1compound(s) with a cell or cell population in vitro or in vivo; (b)measuring one or more of (i) a complex between a binding partner and theformula 1 compound, (ii) proliferation of the cell or cell population,(iii) differentiation of the cell or cell population (iv) an activity ofa protein kinase C, (v) a level of phosphorylation of a protein kinase Csubstrate, (vi) transcription of one or more target genes, (vii)enhancement or inhibition of the cellular response to steroids, e.g.,glucocorticoids, (viii) inhibition of steroid-induced transcription,e.g., glucocorticoids, sex steroids, (ix) inhibition of retrovirus(e.g., HIV, SIV, FIV or SHIV) LTR-driven transcription, or (x)modulation of the numbers of an immune cell population in circulation invivo (e.g., circulating peripheral blood lymphocytes in a mammal such asa primate or a human); and (c) optionally comparing the result obtainedin step (b) with an appropriate control.

26C. The method of embodiment 25C wherein the binding partner is asteroid receptor, a transcription factor or a steroid hormonesuperfamily orphan receptor.

27C. The method of embodiment 25C wherein the biological activitydetermined is a modulating activity of the formula 1 compound forreplication or cytopathic effects associated with a retrovirus, ahepatitis virus or a protozoan parasite.

28C. The method of embodiment 25C wherein the biological activitydetermined is a modulating activity of the formula 1 compound forreplication, cytopathic effects associated with the retrovirus, thehepatitis virus or the protozoan parasite or the biological activitydetermined is metabolism (assay by ³H-thymidine uptake) of a cell orcell population comprising NK cells, phagocytes, monocytes, macrophages,basophils, eosinophils, fibrocytes, transformed cells, virus-infectedcells, bacteria-infected cells or parasite-infected cells.

29C. The method of embodiment 25C wherein the target gene is a virusgene, a bacterial gene, a parasite gene, a gene associated with cancer.

30C. The method of embodiment 29C wherein the virus gene is a polymerasegene, a reverse transcriptase gene, an envelope gene, a protease gene ora gene associated with viral nucleic acid replication or a viralstructural gene.

31C. The method of embodiment 30C wherein the polymerase gene encodes aDNA polymerase or encodes an RNA polymerase.

32C. The method of embodiment 30C wherein the reverse transcriptase geneencodes a human, primate, avian or feline retrovirus reversetranscriptase.

33C. A method comprising administering a compound(s) of formula 1 to ahuman or primate who has a retroviral infection and a CD4 count of 550or less.

34C. The method of embodiment 33C wherein the human has a CD4 count ofabout 20 to about 100 or about 20 to about 80.

35C. The method of embodiment 33C wherein the human has a CD4 count ofabout 30 to about 150.

36C. The method of embodiment 33C wherein the human has a CD4 count ofabout 500 or less, about 450 or less, about 400 or less, about 350 orless, about 300 or less, about 250 or less, about 200 or less, about 150or less, about 100 or less, about 50 or less or about 25 or less orabout 20 or less.

37C. The method of any of embodiments 33C-36C wherein the formula 1compound(s) is present in a composition that comprises one or morenonaqueous liquid excipients and less than about 3% v/v water or any ofthe formulations as disclosed in the specification or any of thenumbered embodiments above.

38C. The method of any of embodiments 33C-37C wherein the formula 1compound(s) is administered according to an intermittent dosing protocolas disclosed in the specification or any of the numbered embodimentsabove.

39C. The method of any of embodiments 30C-45C wherein the human iscoinfected with hepatitis C virus, hepatitis B virus, HSV-1, HSV-2, amalaria parasite, a Pneumocystis parasite, or a Cryptosporidiumparasite.

40C. The method of embodiment 46C wherein level of the HCV is reduced inthe human.

41C. A method comprising administering a formula 1 compound(s) to asubject, or to a nervous system cell(s) in tissue culture whereby theformula 1 compound(s) binds to a receptor associated with a cell(s) inthe nervous system and (1) elicits a biological response in the cell(s)in the nervous system or in the cell(s) in tissue culture and/or (2)elicits a neuronal response that is transmitted to a distant site(s) orcell(s) where the method optionally is used to screen a formula 1compound(s) for its biological activity, to treat a pathologicalcondition (e.g., pathogen infection such as a virus (HIV), a malignancyor a neurological disorder, e.g., AIDS associated dementia, Alzheimer's,Parkinson's, Multiple Sclerosis) in the subject or to determine thebioavailability or metabolism of the formula 1 compound(s) to thesubject or the cell(s) in the nervous system or in tissue culture,wherein metabolism is optionally determined by comparing the biologicaleffect of a formula 1 compound(s) with a control compound, which can bea different formula 1 compound.

42C. The method of embodiment 41 wherein the receptor associated withthe cell in the nervous system is a neurotransmitter receptor(s) (e.g.,a γ-aminobutyric acid receptor such as type A, a NMDA receptor) and/or asteroid receptor (e.g., androgen receptor, estrogen receptor).

43C. The method of embodiment 41C or 42C wherein the cell(s) in thenervous system is a neuron(s), and astrocyte(s) and/or a glial cell(s).

44C. The method of embodiment 41C, 42C or 43C wherein the biologicalresponse in the cell(s) in the nervous system or in the cell(s) intissue culture is increased or decreased transcription of a gene(s)(e.g., a neurotransmitter, vasopressin, a heat shock protein), increasedor decreased secretion of a protein(s) (e.g., vasopressin), reduceddamage from oxidative stress, enhanced nitric oxide release and/orenhanced neurite growth.

45C. The method of any of embodiments 1C-44C wherein the compound(s) offormula 1 is any one or more formula 1 compound selected from thecompounds or one or more of the species of compounds within the generanamed in compound groups 1 through 21-10-6.

46C. A method to (a) modulate (detectably increase or decrease) theexpression of at least one immune cell antigen by an immune cell in asubject, wherein the immune cell antigen is selected from CD3, CD11c,CD14, CD16, CD19, CD25, CD38, CD56, CD62L, CD69, CD45RA, CD45RO, CD123,HLA-DR, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNFα, IGF₁ and γIFN,or (b) activate CD8⁺ T cells or CD8⁻ T cells in a subject, wherein theactivation comprises at least transiently enhanced expression of CD25 orCD69 by the T cells, or (c) increase the proportion of CD8⁺ or CD8⁻lymphokine activated killer cells in a subject's CD16+ cells (e.g.,CD8⁺, CD16⁺, CD38⁺ or cells CD8⁻, CD16⁺, CD38⁺), or (d) increase theproportion of (i) CD8⁻, CD16⁺ natural killer cells, (ii) CD8⁺, CD16⁺natural killer cells or (iii) CD8⁻, CD16⁺ cells that mediateantibody-dependent cell-mediated cytotoxicity, or (iv) CD8⁺, CD16⁺ cellsthat mediate antibody-dependent cell-mediated cytotoxicity, or (e)increase the proportion of dendritic cell precursors in a subject'scirculating white blood cells (e.g., Lin⁻, HLA-DR⁺, CD123⁺ or Lin⁻HLA-DR⁺, CD11c⁺ cells) or (f) increase the proportion of CD45RA⁺ T cellsor CD45⁺, RO⁺ T cells in a subject's circulating white blood cells, or(g) change (increase or decrease) the proportion or relative numbers ofCD62L⁺ T cells in a subject's circulating white blood cells, or (h)increase the proportion of CD8⁺ or CD4⁺ T cells that express CD62L in asubject's circulating CD8⁺ or CD4⁺ T cells, or (i) decrease theproportion of CD8⁺ or CD4⁺ T cells that express CD62L in a subject'scirculating CD8⁺ or CD4⁺ T cells, or (j) increase the proportion ofHLA-DR⁺, CD8⁺, CD38⁺ cells in a subject's circulating white blood cells,or (k) decrease the level of IL-4 or IL-10 that is expressed by orpresent in a subject's white blood cells or in a subject's plasma (orthat is expressed after the subject's white cells are stimulated invitro), (I) at least transiently increase the number of dendritic cellprecursors or dendritic cells that are present in a subject's whiteblood cells or in a subject's plasma, or (m) enhance the capacity of animmune cell, e.g., macrophages, CD4⁺ T cells, CD8⁺ T cells to expressIL-2, IL-12 or γIFN or to activate such cells, the method comprisingadministering to the subject an effective amount of a formula 1compound, which is optionally present in a composition comprising apharmaceutically acceptable excipient.

47C. The method of embodiment 46C wherein formula 1 has the structure

wherein R¹ is —OH or a group (e.g., a C1-30 ester) that canhydrolytically convert under physiological conditions to —OH, either ofwhich may be in the α- or β-configuration; R² is hydrogen in the α- orβ-configuration, or R² is absent when there is a double bond at the 5-6position; R³ is —H or —Br, either of which may be in the α- orβ-configuration; R⁴ is —OH or a group (e.g., a C1-30 ester) that canhydrolytically convert under physiological conditions to —OH, either ofwhich may be in the α- or β-configuration, or R⁴ is ═O and the hydrogenatom bonded to the same carbon is absent; R^(4A) is R⁴, —C(O)—CH³ or—C(O)—(CH₂)₁₋₆—CH³; R⁵ is —H or —OH or a group (e.g., a C1-30 ester)that can hydrolytically convert under physiological conditions to —OH,either of which may be in the α- or β-configuration, or R⁵ is ═O and thehydrogen atom bonded to the same carbon is absent; and the dotted lineat the 5-6 position is an optional double bond, or wherein the formula 1compound has the structure shown in any formula 1 compound named ordescribed herein, including the compounds described in embodiments 1-64and 1A-11A above.

48C. The method of embodiment 46C or 47C wherein the formula 1 compoundis administered to the subject daily over a period from one to about 15days, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore days.

49C. The method of embodiment 48C wherein the expression of the immunecell antigen is detectably modulated for at least about 4-7 days afterthe last administration of the formula 1 compound to the subject, e.g.,for at least 4, 5, 6, 7 or more days.

50C. The method of embodiment 48C or 49C wherein the expression of theimmune cell antigen is detectable at least about 8-90 days after thelast administration of the formula 1 compound, e.g., for at least about8, 10, 12, 15, 20, 25, 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63,65, 70, 75, 77, 80, 84, 85, 90, 91 95, 98, 100 or more days.

51C. The method of any of embodiments 46C-51C wherein the subject has animmunosuppression condition, a pathogen infection or a conditionsassociated with a deficient Th1 immune response or an excessive Th2immune response.

52C. The method of embodiment 51C wherein the pathogen infection is aviral infection, a bacterial infection, a yeast infection, a fungalinfection or a viroid infection, e.g., wherein the pathogen infection isa viral infection such as a DNA virus infection or an RNA virusinfection (e.g., an infection caused by a Hepadnavirus, a Parvovirus, aPapovavirus, an Adenovirus, a Herpesvirus, Retrovirus, a Flavivirus, aTogavirus, a Rhabdovirus, a Picornavirus, a Bunyavirus, a Reovirus, anOrthomyxovirus or a Paramyxovirus, such as a HIV1, HIV2, SIV, SHIV oranother virus described herein or in the cited references).

53C. The method of embodiment 52C wherein the subject has animmunosuppression condition that is associated with or caused by apathogen infection.

54C. The method of any of embodiments 46C-53C wherein the subject is amammal, a human, a primate or a rodent.

55C. The method of any of embodiments 46C-54C wherein about 0.05mg/kg/day to about 20 mg/kg/day is administered parenterally (e.g., byintravenous, subcutaneous, intramuscular, or intramedullary injection),topically, orally, sublingually or bucally to the subject, e.g., about0.1 mg/kg/day, about 0.2 mg/kg/day, about 0.5 mg/kg/day, about 1.0mg/kg/day, about 1.5 mg/kg/day, about 2 mg/kg/day, about 2.5 mg/kg/day,about 3.0 mg/kg/day, about 4 mg/kg/day or about 6 mg/kg/day, i.e., about0.1-10 mg/kg/day, typically about 0.2-7 mg/kg/day.

56C. The method of embodiment 55C wherein the subject is concurrentlytaking one or more second therapeutic agents to treat a pathogeninfection, e.g., a viral infection, such as a HIV-1 infection, a HIV-2infection, a HAV infection, a HBV infection, a HCV infection, an EpsteinBarr virus infection, a HSV-1 infection, a HSV-2 infection, humanherpesvirus 6 infection, human herpesvirus 7 infection, humanherpesvirus 8 infection, or a bacterial infection or a parasiteinfection, such as a malaria infection, Leishmaniasis,Cryptosporidiosis, Toxoplasmosis, a Mycoplasma infection, a Trichomonasinfection, a Chlamidya infection, a Pneumocystis infection, a Salmonellainfection, a Listeria infection, an Escherichia coli infection, aYersinia infection, a Vibrio infection, a Pseudomonas infection, aMycobacterium infection, a Haemophilus infection, a Neisseria infection,a Staphylococcus infection or a Streptococcus infection.

57C. The method of embodiment 56C wherein the one or more secondtherapeutic agents is a protease inhibitor, a reverse transcriptaseinhibitor, a viral, bacterial or parasite DNA or RNA polymeraseinhibitor, an antibacterial antibiotic or an antifungal agent, such asAZT, ddI, ddC, D4T, 3TC, a viral (e.g., HIV) fusion inhibitor,hydroxyurea, nelfinavir, amprenavir, saquinavir, ritonavir, indinavir,chloroquine, a chloroquine analog, amphotericin B, fluconazole,clotrimazole, itraconazole, ketoconazole, isoniazid, dapsone, rifampin,cycloserine, erythromycin, a tetracycline antibiotic, vancomycin,ethambutol, pyrazinamide, a fluororoquinolone (e.g., ciprofloxacin,norfloxacin), a cephalosporin antibiotic, a β-lactam antibiotic or anaminoglycoside antoibiotic (e.g., streptomycin, kanamycin, tobramycin).

58C. The method of any of embodiments 46C-57C wherein the subject is ahuman, a primate, a canine, a feline or a rodent.

59C. A composition comprising an effective amount of an immune cellsubset modulatory compound of formula 1 and a pharmaceuticallyacceptable carrier.

60C. The composition of embodiment 59C wherein the immune cell subset is(1) CD8⁺ T cells, (2) CD4⁺ T cells, (3) CD8⁺ lymphokine activated killercells, (4) CD8⁻ lymphokine activated killer cells, (5) CD8⁻, CD16⁺natural killer cells, (6) CD8⁺, CD16⁺ natural killer cells, (7) CD8⁻,CD16⁺ cells that mediate antibody-dependent cell-mediated cytotoxicity,(8) CD8⁺, CD16⁺ cells that mediate antibody-dependent cell-mediatedcytotoxicity, (9) dendritic cells or dendritic cell precursors, (10)CD45RA⁺ T cells, (11) CD45RO⁺ T cells, (12) CD45RA⁺, CD45RO⁺ T cells,(13) CD8⁺, CD62L T cells, (11) CD4⁺, CD62L⁺ T cells or (14) HLA-DR⁺,CD8⁺, CD38⁺ T cells.

61C. A method to detect a biological response associated with theadministration of a compound of formula 1 to a subject comprising (1)obtaining a sample from the subject, (2) administering the compound offormula 1 to the subject to obtain a treated subject (3) obtaining asecond sample from the treated subject, (4) within 24 hours of obtainingthe sample, analyzing the sample to obtain control information fordetecting the biological response, (5) within 24 hours of obtaining thesecond sample, analyzing the second sample for the presence or absenceof a biological response to obtain experimental information and (6)optionally comparing the control information with the experimentalinformation to detect the presence, absence, relative magnitude orabsolute magnitude of the biological response

62C. The method of embodiment 61C wherein the compound of formula 1further comprises a pharmaceutically acceptable carrier.

63C. The method of embodiment 61C or 62C wherein the biological responseassociated with the administration of the compound of formula 1 to thesubject is modulation of the expression of a cell surface antigen, anincreased absolute or relative number of cells in an immune cell subset,a decreased absolute or relative number of cells in an immune cellsubset or an unchanged absolute or relative number of cells in an immunecell subset.

64C. The method of embodiment 63C wherein the immune cell subset is CD8⁺T cells, CD4⁺ T cells, CD8+lymphokine activated killer cells, CD8⁻,CD16⁺ natural killer cells, circulating dendritic cell precursors,circulating dendritic cells, tissue dendritic cell precursors, tissuedendritic cells, CD8⁺ lymphokine activated killer cells, CD8⁻ lymphokineactivated killer cells, CD8⁻, CD16⁺ natural killer cells, CD8⁺, CD16⁺natural killer cells, CD8⁻, CD16⁺ cells that mediate antibody-dependentcell-mediated cytotoxicity, CD8⁺, CD16⁺ cells that mediateantibody-dependent cell-mediated cytotoxicity, CD45RA⁺ T cells, CD45RA⁺,CD45RO⁺ T cells, CD45RO⁺ T cells, CD8⁺, CD62L T cells, CD4⁺, CD62L⁺ Tcells or HLA-DR⁺, CD8⁺, CD38⁺ T cells, monocytes or macrophages.

65C. The method of embodiment 64C wherein the biological response is atleast transient modulation of an immune cell antigen or an immuneaccessory cell antigen (e.g., an adhesion molecule at the surface ofendothelial cells or a cytokine receptor at the surface of T cells or Bcells).

66C. The method of embodiment 65C wherein the immune cell antigen is aprotein, glycoprotein or cell surface antigen usually or only expressedby lymphoid cells (lymphocytes or white blood cells or their precursors,e.g., T cells, B cells, monocytes, macrophage, LAK cells, NK cells,dendritic cells).

67C. The method of embodiment 65C wherein the immune cell antigen is aCD molecule, an interleukin or a cytokine, optionally selected fromCD16, CD25, CD38, CD62L, CD69, CD45RA, CD45RO, IL-1, IL-2, IL-4, IL-6,IL-8, IL-10, TNFα, IGF₁ and γIFN.

68C. The method of any of embodiments 61C-67C wherein the subject is ahuman, a primate, a canine, a feline or a rodent.

69C. A method to alter the Th1-Th2 balance in a subject comprisingadministering an effective amount a compound of formula 1 to a subjectwhereby the subject's expression or secretion of IL-4 or IL-10 isdetectably modulated.

70C. The method of embodiment 30 wherein the subject's expression orsecretion of IL-4 or IL-10 is decreased and the Th1-Th2 balance in thesubject's Th1 immune responses to an infection or immunosuppressioncondition is detectably enhanced.

71C. The method of any of embodiments 1C-70C, wherein the formula 1compound is a compound named in any of compound groups 1 through54-53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6,or the formula 1 compound is a species in any genus described in any ofcompound groups 1 through54-53-52-51a6-50c27-49c27-48-47-46-45-44-43-42-41-40-39-38-37-36-35-34-33-32-31-30-29-28-27-39-38-37-36-35-34-33-32-31-30-29-28-27-26-25-23-21-17-10-8-6.

72C. A method to prevent or treat an immune disregulation condition in asubject in need thereof comprising administering to the subject, ordelivering to the subject's tissues, an effective amount of a compoundof formula 1, including compounds where, 0, 1, 2 or 3 of R⁷, R⁸ and R⁹are not —CH₂— or —CHR¹⁰— e.g., where R⁷ is —O—, —S— or —NH—, or where R⁸is —O—, —S— or —NH—, or where R⁹ is —O—, —S—, —NH— or ═N— or where allof R⁷, R⁸ and R⁹ are —CH₂— or —CHR¹⁰—.

73C. The method of embodiment 72C where two of R⁷, R⁸ and R⁹ are not—CH₂—, e.g., where R⁷ is —O— and R⁸ is —O—, R⁷ is —O— and R⁹ is —O—, R⁸is —O— and R⁹ is —O—, R⁷ is —O— and R⁸ is —N—, R⁷ is —O— and R⁹ is —NH—or ═N—, R⁸ is —O— and R⁹ is —NH— or ═N—, R⁷ is —O— and R⁸ is —S—, R⁷ is—O— and R⁹ is —S—, R⁸ is —O— and R⁹ is —S—, R⁷ is —NH— and R⁸ is —NH—,R⁷ is —NH— and R⁹ is —NH— or ═N—, R⁸ is —NH— and R⁹ is —NH— or ═N—, R⁷is —NH— and R⁸ is —O—, R⁷ is —NH— and R⁹ is —O—, R⁸ is —NH— and R⁹ is—O—, R⁷ is —NH— and R⁸ is —S—, R⁷ is —NH— and R⁹ is —S—, R⁸ is —NH— andR⁹ is —S—, R⁷ is —S— and R⁸ is —S—, R⁷ is —S— and R⁹ is —S—, R⁸ is —S—and R⁹ is —S—, R⁷ is —S— and R⁸ is —N—, R⁷ is —S— and R⁹ is —NH— or ═N—,R⁸ is —S— and R⁹ is —NH— or ═N—, R⁷ is —S— and R⁸ is —O—, R⁷ is —S— andR⁹ is —O— or R⁸ is —S— and R⁹ is —O—.

74C. The method of embodiment 72C wherein none of R⁷, R⁸ and R⁹ are—CH₂—, e.g., wherein R⁷ is —O—, R⁸ is —O— and R⁹ is —O—, R⁷ is —O—, R⁸is —O— and R⁹ is —NH— or ═N—, R⁷ is —O—, R⁸ is —NH— and R⁹ is —O—, R⁷ is—NH—, R⁸ is —NH— and R⁹ is

—O—, R⁷ is —O—, R⁸ is —O— and R⁹ is —S—, R⁷ is —O—, R⁸ is —S— and R⁹ is—O—, R⁷ is —S—, R⁸ is —S— and R⁹ is —O—, R⁷ is —NH—, R⁸ is —NH— and R⁹is —NH— or ═N—, R⁷ is —O—, R⁸ is —NH— and R⁹ is —NH— or ═N—, R⁷ is —NH—,R⁸ is —O— and R⁹ is —NH— or ═N—, R⁷ is —O—, R⁸ is —O— and R⁹ is —NH— or═N—, R⁷ is —S—, R⁸ is —NH— and R⁹ is —NH— or ═N—, R⁷ is —NH—, R⁸ is —S—and R⁹ is —NH— or ═N—, R⁷ is —S—, R⁸ is —S— and R⁹ is —NH— or ═N—, R⁷ is—S—, R⁸ is —S— and R⁹ is —S—, R⁷ is —O—, R⁸ is —S— and R⁹ is —S—, R⁷ is—S—, R⁸ is —O— and R⁹ is —S—, R⁷ is —O—, R⁸ is —S— and R⁹ is —S—, R⁷ is—NH—, R⁸ is —S— and R⁹ is —S—, R⁷ is —S—, R⁸ is —NH— and R⁹ is —S—, R⁷is —S—, R⁸ is —NH— and R⁹ is —O—, R⁷ is —NH—, R⁸ is —S— and R⁹ is —O—,R⁷ is —S—, R⁸ is —O— and R⁹ is —NH— or ═N—, R⁷ is —O—, R⁸ is —S— and R⁹is —NH— or ═N—, R⁷ is —NH—, R⁸ is —O— and R⁹ is —S—, or R⁷ is —O—, R⁸ is—NH— and R⁹ is —S—.

75C. The method of any of embodiments 72C through 74C wherein the thereare no double bonds in the formula 1 compound, R¹, R⁵ and R⁶ are in thep configuration, one R⁴ is hydrogen and the immune disregulationcondition is caused by or is associated with inflammation, an autoimmunecondition, an organ or tissue transplant rejection, an infection or itstreatment, a cancer or its treatment, a chemotherapy, a radiationtherapy, trauma, surgery, an allergy condition or an insufficient Th1immune response.

76C. The method of embodiment 75C, wherein the formula 1 compound is acompound named in any of compound groups 1 through 54, or the formula 1compound is a species in any genus described in any of compound groups 1through 54.

Invention embodiments include stimulation of hemopoiesis in a subject.The following embodiments exemplify various aspects if these embodiments

1D. A method to enhance hemopoiesis in a subject in need thereofcomprising administering to the subject, or delivering to the subject'stissues, an effective amount of a compound of formula 1

wherein, each R¹, R², R³, R⁴, R⁵, R⁶ and R¹⁰ independently are —H,—OR^(PR), —SR^(PR), —N(R^(PR))₂, —O—Si—(R¹³)₃, —CHO, —CHS, —CH═NH, —CN,—SCN, —NO₂, —OSO₃H, —OPO₃H, an ester, a thioester, a thionoester, aphosphoester, a phosphothioester, a phosphonoester, a phosphiniester, asulfite ester, a sulfate ester, an amide, an amino acid, a peptide, anether, a thioether, an acyl group, a thioacyl group, a carbonate, acarbamate, an acetal, a thioacetal, a halogen, an optionally substitutedalkyl group, an optionally substituted alkenyl group, an optionallysubstituted alkynyl group, an optionally substituted aryl moiety, anoptionally substituted heteroaryl moiety, an optionally substitutedheterocycle, an optionally substituted monosaccharide, an optionallysubstituted oligosaccharide, a nucleoside, a nucleotide, anoligonucleotide, a polymer, or,

one more of R¹, R², R³, R⁴, R⁵, R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸ are ═O, ═S,═N—OH or ═CH₂ and the hydrogen atom or the second variable group that isbonded to the same carbon atom is absent, or,

all R³ and R⁴ together comprise a structure of formula 2

R⁷ is —C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—C(R¹⁰)₂—,—C(R¹⁰)₂—O—C(R¹⁰)₂—, —C(R¹⁰)₂—S—C(R¹⁰)₂—, —C(R¹⁰)₂—NR^(PR)—C(R¹⁰)₂—,—O—, —O—C(R¹⁰)₂—, —S—, —S—C(R¹⁰)₂—, —NR^(PR)— or —NR^(PR)—C(R¹⁰)₂—,including —CHR¹⁰—, —CHR¹⁰—CHR¹⁰—, —CHR¹⁰—CHR¹⁰—CHR¹⁰—, —CHR¹⁰—O—CHR¹⁰—,—CHR¹⁰—S—CHR¹⁰—, —CHR¹⁰—NR^(PR)—CHR¹⁰—, —O—, —O—CHR¹⁰, —S—, —S—CHR¹⁰—,—NR^(PR)— or —NR^(PR)—CHR¹⁰—

R⁸ and R⁹ independently are —C(R¹⁰)₂—, —C(R¹⁰)₂—C(R¹⁰)₂—, —O—,—O—C(R¹⁰)₂—, —S—, —S—C(R¹⁰)₂—, —NR^(PR)— or —NR^(PR)—C(R¹⁰)₂—, including—CHR¹⁰—, —CHR¹⁰—CHR¹⁰—, —O—, —O—CHR¹⁰—, —S—, —S—CHR¹⁰—, —NR^(PR)— or—NR^(PR)—CHR¹⁰—, or one or both of R⁸ or R⁹ independently are absent,leaving a 5-membered ring;

R¹³ independently is C₁₋₆ alkyl;

R¹⁶ independently are —CH₂—, —O—, —S— or —NH—;

D is a heterocycle or a 4-, 5-, 6- or 7-membered ring that comprisessaturated carbon atoms, wherein 1, 2 or 3 ring carbon atoms of the 4-,5-, 6- or 7-membered ring are optionally independently substituted with—O—, —S— or —NR^(PR)— or where 1, 2 or 3 hydrogen atoms of theheterocycle or where 1 or 2 hydrogen atoms of the 4-, 5-, 6- or7-membered ring are substituted with —OR^(PR), —SR^(PR), —N(R^(PR))₂,—O—Si—(R¹³)₃, —CN, —NO₂, an ester, a thioester, a thionoester, aphosphoester, a phosphothioester, a sulfite ester, a sulfate ester, anamide, an amino acid, a peptide, an ether, a thioether, an acyl group, athioacyl group, a carbonate, a carbamate, an acetal, a thioacetal, ahalogen, an optionally substituted alkyl group, an optionallysubstituted alkenyl group, an optionally substituted alkynyl group, anoptionally substituted aryl moiety, an optionally substituted heteroarylmoiety, an optionally substituted monosaccharide, an optionallysubstituted oligosaccharide, a nucleoside, a nucleotide, anoligonucleotide or a polymer, or,

one more of the ring carbons are substituted with ═O or ═S,

or D comprises two 5- or 6-membered rings, wherein the rings are fusedor are linked by 1 or 2 bonds, or a metabolic precursor or abiologically active metabolite thereof, optionally provided that thecompound is not 5-androstene-3β-ol-17-one, 5-androstene-3β,17β-diol,5-androstene-3β,7β,17β-triol or a derivative of any of these threecompounds that can convert to these compounds by hydrolysis

2D. The method of embodiment 1D wherein the subject's circulatingplatelets, red cells, mature myelomonocytic cells, or their precursorcells, in circulation or in tissue is detectably increased.

3D. The method of embodiment 2D wherein the subject's circulatingplatelets are detectably increased.

4D. The method of embodiment 3D wherein the method optionally furthercomprises administration of an effective amount of G-CSF, GM-CSF, IL-3,IL-6, IL-11, erythropoietin or thrombopoietin.

5D. The method of embodiment 2D wherein the subject's circulatingmyelomonocytic cells are detectably increased.

6D. The method of embodiment 2D wherein the circulating myelomonocyticcells are neutrophils.

7D. The method of embodiment 2D wherein the method further comprisesadministration of an effective amount of G-CSF, GM-CSF, M-CSF, IL-3,IL-5 or IL-6.

8D. The method of embodiment 2D wherein the myelomonocytic cells arebasophils, neutrophils or eosinophils.

9D. The method of embodiment 2D wherein the subject's circulating redcells are detectably increased.

10D. The method of embodiment 9D wherein the subject is has renalfailure.

11D. The method of embodiment 9D wherein the method further comprisesadministration of an effective amount of G-CSF, GM-CSF, IL-3, IL-6 orerythropoietin.

12D. The method of embodiment 2D wherein the formula 1 compound ispresent in a composition comprising an acceptable carrier and the methodoptionally further comprises administration of a neutrophil or monocytestimulator.

13D. The method of embodiment 12D wherein neutrophil or monocytestimulator is a TNF, a lithium salt, deuterium oxide, levamisole,lactoferrin, thyroxine, triiodothyromine, anthrax toxin, ascorbic acid,1-palmitoyl-lysophosphatidic acid, a calcium ionophore, cytochalasin B,sodium butyrate, piracetamine, micronized L-arginine, hydroxyurea or abacterial lipopolysaccharide.

14D. The method of embodiment 2D further comprising the steps ofobtaining blood from the subject before administration of the formula 1compound and measuring the subject's white or red cell counts andoptionally, on one, two, three or more occasions, measuring thesubject's circulating white cell counts after administration of theformula 1 compound.

15D. The method of embodiment 2D wherein the formula 1 compound is acompound named in any of the compound groups disclosed herein.

16D. The method of embodiment 1D wherein the subject has, or is subjectto developing, thrombocytopenia or neutropenia.

17D. The method of embodiment 16D wherein the subject hasthrombocytopenia or neutropenia.

18D. The method of embodiment 1D wherein about 0.05 mg to about 30 mg ofthe formula 1 compound is administered per kg of the subject's weightper day.

19D. The method of any of embodiments 1D-18D wherein the compound offormula 1 has formula 3

20D. The method of embodiment 19D wherein R¹ is —OH, alkoxy or an ester,R² is —OH, ═O, alkoxy or an ester, R³ is —H, —OH, alkoxy, an ester or ahalogen, one R⁴ is —H or it is absent and the other R⁴ is —OH, ═O, —SH,—C(O)—CH₃, alkoxy or an ester and wherein R¹, R² and R³ areindependently in the α or the β configuration when they are not ═O.

21D. The method of embodiment 20D wherein there is no double bondpresent in the molecule.

22D. A method to enhance thrombopoiesis, myelopoiesis or erythropoiesisin a subject comprising administering to the subject, or delivering tothe subject's tissues, an effective amount of the compound of embodiment1.

23D. The method of embodiment 22D wherein the subject has or is subjectto thrombocytopenia or neutropenia.

24D. The method of embodiment 23D wherein the subject hasthrombocytopenia or neutropenia.

25D. The method of embodiment 22D wherein the subject is a human.

26D. The method of embodiment 22D wherein the formula 1 compound is acompound named in any of the compound groups disclosed herein.

27D. The method of embodiment 22D wherein about 0.05 mg to about 30 mgof the formula 1 compound is administered per kg of the subject's weightper day.

28D. The method of embodiment 27D wherein the formula 1 compound ispresent in a composition that comprises a pharmaceutically acceptablecarrier.

29D. The method of embodiment 28D wherein the pharmaceuticallyacceptable carrier is deuterium oxide, which comprises at least about20% v/v of the water in the composition.

30D. The method of embodiment 19D wherein the subject's myeloperoxidaseindex is enhanced.

31D. The method of embodiment 30D wherein the formula 1 compound ispresent in a composition that comprises one or more pharmaceuticallyacceptable carriers, which optionally include a halogen salt.

32D. A method to treat a blood cell deficiency in a subject comprisingadministering to the subject, or delivering to the subject's tissues, aneffective amount of a compound of formula 1.

33D. The method of embodiment 32D wherein the formula 1 compound has thestructure

wherein one, two or three of R⁷, R⁸ and R⁹ are —CH₂— or —CH═ and whereinthe configuration of hydrogen atoms at the 5 (if present), 8, 9 and 14positions respectively are α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α,α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β,β.β.α.β, β.β.β.α or β.β.β.β, typically α.α.β.α or β.α.β.α.

34D. The method of embodiment 33D wherein the formula 1 compound has thestructure

35D. The method of embodiment 34D wherein R¹, R² and R⁴ independentlyare —OH, —SCN, a C2-C20 ester or C1-C20 alkoxy, R³ is —H and two orthree of R⁷, R⁸ and R⁹ are —CH₂—.

36D. The method of embodiment 34D or 35D wherein the formula 1 compoundhas the structure

37D. The method of any of embodiments 33D-36D wherein the configurationof hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are α.α.β.α or β.α.β.α.

38D. The method of embodiment 32D wherein the formula 1 compound has thestructure

wherein one R⁴ is absent when there is a double bond at the 16-17position and wherein R⁷, R⁸ and R⁹ are independently selected andwherein one, two or three of R⁷, R⁸ and R⁹ are not —CH₂— or —CH═ andwherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are in the α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α,α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β,β.β.α.β, β.β.β.α or β.β.β.β, configurations, typically α.α.β.α orβ.α.β.α.

39D. The method of embodiment 38D wherein R⁸ is —CH₂—, —O—, —S— or —NH—.

40D. The method of embodiment 38D or 39D wherein R⁷ is —CH₂—CHR¹⁰—,—CH₂—, —O—CHR¹⁰— or —O—C(O)—.

41D. The method of embodiment 38D, 39D or 40D wherein R⁸ or R⁹ isabsent.

42D. The method of embodiment 38D or 39D wherein R⁷ and R⁹ independentlyare —CHR¹⁰—, —CH₂—, —CH═, —O—, —S— or —NH—, wherein R¹⁰ is —OH, —SH, aC₁₋₃₀ organic moiety, a C₁₋₃₀ ester, C₁₋₁₀ optionally substituted alkyl,C₁₋₁₀ optionally substituted alkoxy, C₁₋₁₀ optionally substitutedalkenyl or C₁₋₁₀ optionally substituted alkynyl.

43D. The method of embodiment 32D wherein the formula 1 method has thestructure

wherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are in the α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α,α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β,β.β.α.β, β.β.β.α or β.β.β.β configurations, typically α.α.β.α orβ.α.β.α.

44D. The method of embodiment 43D wherein R⁴ is —OH, ═O, —SH, —SCN, aC₁₋₃₀ ester or C₁₋₃₀ alkoxy, wherein the ester or alkoxy moiety isoptionally substituted with one, two or more independently selectedsubstituents, which are optionally selected from —F, —Cl, —Br, —I, —O—,═O, —S—, —NH—, —R^(PR), —OR^(PR), —SR^(PR) or —NHR^(PR).

45D. The method of embodiment 43D or 44D wherein R¹ is —OH, ═O, —SH,—SCN, a C₁₋₃₀ ester or C₁-C₃₀ alkoxy, wherein the ester or alkoxy moietyis optionally substituted with one, two or more independently selectedsubstituents, which are optionally selected from —F, —Cl, —Br, —I, —O—,═O, —S—, —NH—, —R^(PR), —OR^(PR), —SR^(PR) or —NHR^(PR).

46D. The method of any of embodiments 32D-45D wherein a second R¹ ispresent and it is a moiety other than hydrogen, e.g., —OH, —SH, —SCN, aC₁₋₃₀ ester, C₁₋₃₀ alkoxy, C₁₋₃₀ alkynyl or a monosaccharide wherein theester, alkoxy, alkynyl or monosaccharide is optionally substituted withone, two or more independently selected substituents, which areoptionally selected from —F, —Cl, —Br, —I, —O—, ═O, —S—, —NH—, —R^(PR),—OR^(PR), —SR^(PR) or —NHR^(PR).

47D. The method of any of embodiments 32D-46D wherein a second R² ispresent and it is a moiety other than hydrogen, e.g., —OH, —SH, —SCN, aC₁₋₃₀ ester or C₁₋₃₀ alkoxy, C₁₋₃₀ alkynyl or a monosaccharide whereinthe ester, alkoxy, alkynyl or monosaccharide is optionally substitutedwith one, two or more independently selected substituents, which areoptionally selected from —F, —Cl, —Br, —I, —O—, ═O, —S—, —NH—, —R^(PR),—OR^(PR), —SR^(PR) or —NHR^(PR).

48D. The method of any of embodiments 32D-47D wherein a second R³ ispresent and it is a moiety other than hydrogen, e.g., —OH, —SH, —SCN, aC₁₋₃₀ ester or C₁₋₃₀ alkoxy, C₁₋₃₀ alkynyl or a monosaccharide whereinthe ester, alkoxy, alkynyl or monosaccharide is optionally substitutedwith one, two or more independently selected substituents, which areoptionally selected from —F, —Cl, —Br, —I, —O—, ═O, —S—, —NH—, —R^(PR),—OR^(PR), —SR^(PR) or —NHR^(PR).

49D. The method of any of embodiments 32D-48D wherein a second R⁴ ispresent and it is a moiety other than hydrogen, e.g., —OH, —SH, —SCN, aC₁₋₃₀ ester or C₁₋₃₀ alkoxy, C₁₋₃₀ alkynyl or a monosaccharide whereinthe ester, alkoxy, alkynyl or monosaccharide is optionally substitutedwith one, two or more independently selected substituents, which areoptionally selected from —F, —Cl, —Br, —I, —O—, ═O, —S—, —NH—, —R^(PR),—OR^(PR), —SR^(PR) or —NHR^(PR).

50D. The method of any of embodiments 32D-49D wherein there is a doublebond at the 1-2 position.

51D. The method of any of embodiments 32D-49D wherein there is a doublebond at the 4-5 position.

52D. The method of any of embodiments 32D-49D wherein there is a doublebond at the 5-6 position.

53D. The method of any of embodiments 32D-49D wherein there is a doublebond at the 16-17 position.

54D. The method of any of embodiments 32D-49D wherein there are doublebonds at the 1-2 and 4-5 positions.

55D. The method of any of embodiments 32D-49D wherein there are doublebonds at the 1-2 and 5-6 positions.

56D. The method of any of embodiments 32D-49D wherein there are doublebonds at the 1-2 and 16-17 positions.

57D. The method of any of embodiments 32D-49D wherein there are doublebonds at the 4-5 and 16-17 positions.

58D. The method of any of embodiments 32D-49D wherein there are doublebonds at the 5-6 and 16-17 positions.

59D. The method of any of embodiments 32D-49D wherein there are doublebonds at the 1-2, 4-5 and 16-17 positions.

60D. The method of any of embodiments 32D-49D wherein there are doublebonds at the 1-2, 5-6 and 16-17 positions.

61D. A compound of formula 1, e.g., a compound in any compound group orembodiment disclosed herein.

62D. A composition comprising a compound of formula 1, e.g., a compoundin any compound group or embodiment disclosed herein, and an excipient.

63D. Use of a compound of formula 1, e.g., a compound in any compoundgroup or embodiment disclosed herein, to manufacture a medicament forthe treatment of a blood cell deficiency, e.g., NP of TP, in a subject,e.g., a mammal or a human.

64D. A product produced by the process of contacting a formula 1compound, e.g., a compound in any compound group or embodiment disclosedherein, and an excipient.

65D. A kit comprising a formulation that comprises a unit dosage or amultiple dosage comprising a formula 1 compound, e.g., a compound in anycompound group or embodiment disclosed herein, and one or moreexcipients wherein the formulation is dispensed in a suitable container,wherein the kit further comprises a label that provides informationabout one or more of (1) the formula 1 compound's chemical structure,(2) any recommended dosing regimen, (3) any adverse effects ofadministering the formula 1 compound to a subject that are required tobe disclosed and (4) the amount of the formula 1 compound that ispresent in each unit dose or in the entire container.

Embodiments related to BrEA hemihydrate include the following.

1E. 16α-Bromo-3β-hydroxy-5α-androstan-17-one hemihydrate substantiallyfree of other forms of 16α-bromo-3β-hydroxy-5α-androstan-17-one.

2E. The 16α-bromo-3β-hydroxy-5α-androstan-17-one hemihydratesubstantially free of other forms of16α-bromo-3β-hydroxy-5α-androstan-17-one of embodiment 1E wherein16α-bromo-3β-hydroxy-5α-androstan-17-one hemihydrate comprises at leastabout 55% w/w of the 16α-bromo-3β-hydroxy-5α-androstan-17-one that ispresent.

3E. The 16α-bromo-3β-hydroxy-5α-androstan-17-one hemihydratesubstantially free of other forms of16α-bromo-3β-hydroxy-5α-androstan-17-one of embodiment 1E wherein16α-bromo-3β-hydroxy-5α-androstan-17-one hemihydrate comprises at leastabout 98% w/w of the 16α-bromo-3β-hydroxy-5α-androstan-17-one that ispresent.

4E. The 16α-bromo-3β-hydroxy-5α-androstan-17-one hemihydrate ofembodiment 3E, characterized by one or more of (1) an absorptionendotherm onset as measured by differential scanning calorimetryanalysis of about 100° C., (2) two carbonyl absorption bands at about1741 cm⁻¹ and 1750 cm⁻¹ as measured by Fourier transform infraredabsorption spectroscopy, (3) a water content of about 2.4% w/w to about2.6% w/w as measured by Karl Fisher titration and (4) 1, 2, 3, 4, 5, 6or more of the X-ray powder diffraction peaks at 17.8, 23.8, 24.2,26.9-27.2, 28.6, 30.1 or 32.2 Theta, obtained from an XRD spectrum of16α-bromo-3β-hydroxy-5α-androstan-17-one hemihydrate using Cu—Kαradiation.

5E. The 16α-bromo-3β-hydroxy-5α-androstan-17-one of embodiment 1E, 2E or3E in a composition comprising an excipient suitable for humanpharmaceutical use or for veterinary use.

6E. A method to make 16α-bromo-3β-hydroxy-5α-androstan-17-onehemihydrate comprising contacting water,16α-bromo-3β-hydroxy-5α-androstan-17-one and a C₁₀₆ alcohol.

7E. The method of embodiment 6E wherein the C1-C6 alcohol is ethanol.

8E. The method of embodiment 6E wherein the solution comprises about5-25% w/w water, about 30-45% w/w ethanol and about 30-45% w/w of a16α-bromo-36-hydroxy-5α-androstan-17-one preparation.

9E. The method of embodiment 6E wherein the solution comprises about18-22% w/w water, about 37-43% w/w ethanol and about 37-43% w/w of a16α-bromo-36-hydroxy-5α-androstan-17-one preparation.

10E. The method of embodiment 9E wherein the solution is at atemperature of about −20° C. to about 45° C.

11E. The 16α-bromo-3β-hydroxy-5α-androstan-17-one hemihydrate ofembodiment 1E substantially free of anhydrous16α-bromo-3β-hydroxy-5α-androstan-17-one or substantially free ofamorphous 16α-bromo-3β-hydroxy-5α-androstan-17-one.

12E. A product produced by the process of contacting a solutioncomprising water, 16α-bromo-3β-hydroxy-5α-androstan-17-one and a C1-C6alcohol.

13E. The product of embodiment 12E that is16α-bromo-3β-hydroxy-5α-androstan-17-one hemihydrate.

14E. The product of embodiment 12E wherein the solution comprises about5-25% w/w water, about 30-45% w/w ethanol and about 30-45% w/w of a16α-bromo-3β-hydroxy-5α-androstan-17-one preparation.

Embodiments related to buccal and sublingual formulations that comprisea formula 1 compound include the following.

1F. A solid buccal, sublingual or transmucosal oral mucosa formulation(or composition) having dimensions, e.g., as disclosed herein, which fitinto the buccal cavity or under the tongue of a subject, comprising (1)a suitable amount of any formula 1 compound, e.g., about 0.1 to about100 mg or about 0.5-50 mg, or about 20-30 mg disclosed herein, (2) about20 to about 75 percent by weight of a PEG having a molecular weight offrom about 100-4000, (3) about 2 to about 54 percent by weight of a PEGhaving a molecular weight of from about 6000-20,000, and (4) about 1 toabout 40 percent by weight of polyethylene oxide having a molecularweight of from about 100,000 to about 5,000,000. The formulation maycomprise about 10, 20, 25, 30 or 40 mg of the formula 1 compound.

2F. The formulation of embodiment 1 wherein comprising a formulationthat substantially or completely erodes or disintegrates over a periodof about 10-30 minutes while in water at about 37° C. or in a subject'soral cavity.

3F. The formulation of embodiments 1F or 2F wherein the formula 1compound is homogeneously or nonhomogenously dispersed in anon-crystalline, solidified polymeric matrix which adheres to oralmucosa after being activated by or contacting sufficient water orsaliva.

4F. The formulation of embodiments 1F-3F wherein the PEG of component(2) is a mixture of low molecular weight polyethylene glycols, whichoptionally comprises 1, 2 or more of PEG1000, PEG1450 and PEG3350.

5F. The formulation of embodiments 1F-4F wherein the PEG of component(2) is present in a proportion of from about 35% to about 65% by weightof the total composition.

6F. The formulation of embodiments 1F-5F wherein the PEG of component(3) comprises PEG8000.

7F. The formulation of embodiments 1F-6F wherein the PEG of component(3) comprises (i) PEG20000 or (ii) a mixture of PEGs, e.g., a mixture ofPEG8000 and PEG20,000.

8F. The formulation of embodiments 1F-7F wherein the PEG of component(4) comprises polyethylene oxide having a molecular weight of about100,000 (which is optionally present in an amount from about 2% to about10% by weight) or a molecular weight of about 5,000,000.

9F. The formulation of embodiments 1F-8F wherein the formulation furthercomprises a plasticizer, e.g., propylene glycol, which is optionallypresent in a proportion of about 3 percent by weight.

10F. A method to prevent, treat, ameliorate or slow the progression ordevelopment of a condition or symptom disclosed herein in a subjectcomprising administering to the subject an effective amount of theformulation of embodiments 1F-9F, e.g., using a dosing protocolessentially as described herein, e.g., by continuous daily dosing or byintermittent administration. Related embodiments provide the use of theformulation of embodiments 1F-9F to prepare a medicament to prevent,treat, ameliorate or slow the progression of a condition or symptomdisclosed herein, e.g., by continuous daily dosing or by intermittentadministration. Suitable subjects include mammals, humans or subjectsdisclosed herein.

11F. The method of embodiment 10F wherein the formula 1 compound is acompound disclosed in any of the compound groups contained herein, e.g.,compound groups 1-54, or any formula 1 structure disclosed herein.

12F. A method for administering a formula 1 compound to a subject inneed of treatment comprising contacting the mucosa of the subject aneffective amount of a composition comprising a formula 1 compound and acyclodextrin. The composition may comprise a formulation that comprisesabout 0.1 to about 100 mg, e.g., about 10, 20, 25, 30 or 40 mg of theformula 1 compound per unit dosage. Suitable subjects include mammals,humans or subjects disclosed herein. The subject may have or be subjectto developing a condition or symptom disclosed herein and the method isused to prevent, treat, ameliorate or slow the progression ordevelopment of the condition or symptom. The formulation optionallycomprises a cyclodextrin-formula 1 compound complex.

13F. The method of embodiment 12F wherein the cyclodextrin isγ-cyclodextrin, β-cyclodextrin or hydroxypropyl-β-cyclodextrin, any ofwhich are crystalline, amorphous or a mixture thereof.

14F. The method of embodiments 12F and 13F wherein the formulationfurther comprises propylene oxide or epichlorohydrin, which areoptionally prepared as condensation products with the cyclodextrin.

15F. The method of embodiments 12F-14F wherein the formula 1 compound isa compound disclosed in any of the compound groups contained herein,e.g., compound groups 1-54, or any formula 1 structure disclosed herein.

16F. A method for administering a formula 1 compound to a subject inneed of treatment comprising contacting the mucosa of the subject withan effective amount of a composition comprising a formula 1 compound andabout 70-90% w/w (e.g., about 80-85%) of a low molecular weight PEG (amol. weight of about 200 to about 2000, e.g., PEG1000), about 0-4% w/w(e.g., about 0.5%) of a higher molecular weight PEG (a mol. weight ofabout 3000 to about 20000, e.g., PEG3350 or PEG 8000), about 0-4% w/w(e.g., about 0.5%) of a long chain saturated or unsaturated carboxylicacid (e.g., a 010-24 carboxylic acid or C12-18 such as myristic acid),about 0.1-4% w/w of polyethylene oxide (e.g., about 0.5%) (mol. weightabout 100,000-5,000,000) and about 10-20% w/w colloidal silica (e.g.,about 12-18% or about 16%). The composition may comprise a formulationthat comprises about 0.1 to about 100 mg, e.g., about 10, 20, 25, 30 or40 mg of the formula 1 compound per unit dosage. Suitable subjectsinclude mammals, humans or subjects disclosed herein. The subject mayhave or be subject to developing a condition or symptom disclosed hereinand the method is used to prevent, treat, ameliorate or slow theprogression or development of the condition or symptom.

17F. A method for administering a formula 1 compound to a subject inneed of treatment comprising contacting the mucosa of the subject withan effective amount of a composition comprising a formula 1 compound,erythritol, a crystalline cellulose, a crospovidone and optionallymannitol. The composition may comprise a formulation that comprisesabout 0.1 to about 100 mg, e.g., about 10, 20, 25, 30 or 40 mg of theformula 1 compound per unit dosage. Suitable subjects include mammals,humans or subjects disclosed herein. The subject may have or be subjectto developing a condition or symptom disclosed herein and the method isused to prevent, treat, ameliorate or slow the progression ordevelopment of the condition or symptom.

18F. The method of embodiment 17F wherein the composition comprises asolid formulation and the erythritol is comprises a proportion of 5-90parts by weight, based on 100 parts by weight of the composition.

19F. The method of embodiments 17F or 18F wherein the crystallinecellulose comprises a proportion of 3-50 parts by weight, based on 100parts by weight of the composition.

20F. The method of embodiments 17F-19F wherein the crospovidonecomprises a proportion of 1-10 parts by weight of the composition.

21F. The method of embodiments 17F-20F wherein the formula 1 compound,the erythritol, the crystalline cellulose, the crospovidone and theoptional mannitol, are uniformly mixed.

22F. The method of embodiments 17F-20F wherein the composition comprisesa tablet or lozenge.

23F. The method of embodiments 17F-20F wherein the composition comprisesabout 0.3-50 parts by weight of the formula 1 compound, about 50-80parts by weight of erythritol, about 5-20 parts by weight of crystallinecellulose and about 3-7 parts by weight of crospovidone. Embodimentsrelated to treatment of the delayed effects of radiation include thefollowing.

1G. A method to treat a symptom or condition associated with one or moredelayed adverse or unwanted effects of radiation exposure in a subjectin need thereof comprising administering to the subject, or deliveringto the subject's tissues, an effective amount of a compound of formula1, wherein the formula 1 compound is administered or delivered to thesubject's tissues beginning at least 1 day (e.g., at 2, 4, 6, 7, 14, 30or more days) after the subject has been exposed to a dose of radiationthat will cause or could potentially cause the one or more delayedadverse or unwanted effects of the radiation exposure, or wherein theformula 1 compound is administered or delivered to the subject's tissuesbeginning at least 1 day (e.g., at 2, 4, 6, 7, 14, 30 or more days)after the subject has been exposed to at least one subdose of a plannedcourse of radiation exposures that will cause or could potentially causethe one or more delayed adverse effects or unwanted effects of theradiation exposure.

2G. The method of embodiment 1G wherein the subject has received a totalradiation dose of at least about 0.5 Gy to about 300 Gy, wherein thesubject received the radiation dose in a single dose or in two or moredivided doses.

3G. The method of embodiment 1G or 2G wherein the subject has received atotal radiation dose of at least about Gy 1 to about 200 Gy.

4G. The method of embodiment 1G, 2G or 3G wherein the subject hasreceived a total radiation dose of at least about Gy 2 to about 150 Gy.

5G. The method of embodiment 1G, 2G, 3G, or 4G wherein the symptom orcondition associated with one or more delayed adverse effect ofradiation is one or more of encephalopathy, myelopathy, nausea,vomiting, diarrhea, acute inflammation, chronic inflammation, edema,pain, headache, depression, fever, malaise, weakness, hair loss, skinatrophy, skin ulceration, skin lesion, keratosis, telangiectasia,infection, hypoplasia, atrophy, fibrosis, pneumonitis, bone marrowhypoplasia, hemorrhage or cytopenia.

6G. The method of embodiment 5G wherein the infection is a bacterial,viral, fungal, parasite or yeast infection, or wherein the fibrosis islung fibrosis or wherein the cytopenia is anemia, leukopenia orthrombocytopenia.

7G. The method of embodiment 1G, 2G, 3G, or 4G wherein the symptom orcondition associated with one or more delayed adverse or unwanted effectof the radiation exposure is caused by or associated with radiationdamage to one or more of bone marrow cells, bowel epithelium, bonemarrow, testicles, ovaries, brain nerves or tissue, peripheral nerves,spinal cord nerves or tissue or skin epithelium.

8G. The method of embodiment 1G to 7G wherein the subject has receivedor will receive a total radiation dose of at least about 0.5 Gy, atleast about 2 Gy, at least about 4 Gy, at least about 6 Gy or at leastabout 10 Gy.

9G. The method of embodiment 1G to 7G wherein the subject has receivedor is anticipated to receive a total radiation dose of at least about 10Gy, e.g., about 10, 20, 30, 40, 50, 100, 150, 200 or 300 Gy.

10G. The method of embodiment 1G to 9G wherein the formula 1 compound isadministered to the subject or delivered to the subject's tissues bydaily administration of the formula 1 compound or the formula 1 compoundis administered to the subject or delivered to the subject's tissuesusing an intermittent dosing protocol or method and wherein theadministration to the subject or the delivery to the subject's tissuesis conducted for a period of at least about 2 days to at least about 2years.

11G. The method of embodiment 1G to 10G wherein about 0.05 mg/kg/day toabout 500 mg/kg/day of the formula 1 compound is administered to thesubject or delivered to the subject's tissues.

12G. The method of embodiment 11G wherein about 0.1 mg/kg/day to about50 mg/kg/day of the formula 1 compound is administered to the subject ordelivered to the subject's tissues.

13G. The method of embodiment 12G wherein about 0.4 mg/kg/day to about25 mg/kg/day of the formula 1 compound is administered to the subject ordelivered to the subject's tissues.

14G. The method of any of embodiments 1G to 13G wherein the all or someof the formula 1 compound is administered to the subject or delivered tothe subject's tissues by one or more of an oral, parenteral, topical,aerosol, nasal, rectal, vaginal, buccal or sublingual route.

15G. The method of any of embodiments 1G to 14G wherein the formula 1compound has the formula 20

wherein R¹ is —H, —OH, ═O, —SH, ═S, —OCH₃, —OC₂H₅, —O—S(O)(O)—O—Na⁺,—O—S(O)(O)—OC₂H₅, —CH₃, —C₂H₅, —OC(O)C(CH₃)₃, —OC(O)CH₃, an optionallysubstituted monosaccharide, an optionally substituted oligosacccharidecomprising two, three or more covalently linked optionally substitutedmonosaccharides, or an amino acid;

R² is —H, —OH, ═O, —CH₃, —CF₃, —OCH₃, —OC₂H₅, —C₂H₅, —OCH₂CH₂CH₃,—OCH₂CH₂CH₂CH₃, —F, —Cl, —Br or —I;

R³ is —H, —F, —Cl, —Br, —I, —OH, —SH, ═O, ═CH₂, —NH₂, —CH₃, —CF₃, —C₂H₅,—O—C(O)—CH₃, —O—C(O)—CH₂CH₃, —O—C(O)—CH₂CH₂CH₃, —C(O)—CH₃, —C(O)—CH₂CH₃,—C(O)—CH₂CH₂CH₃;

R⁴ is —H, —F, —Cl, —Br, —I, —OH, ═O, ═CH₂, —CCH, —SH—O—C(O)—CH₃,—O—C(O)—CH₂CH₃, —O—C(O)—CH₂CH₂CH₃, —C(O)—CH₃, —C(O)—CH₂CH₃,—C(O)—CH₂CH₂CH₃, —CHOH—CH₃, —CHOH—CH₂CH₃, —CHOH—CH₂CH₂CH₃, —CHOH—C₆H₁₃,an optionally substituted monosaccharide, an optionally substitutedoligosacccharide comprising two, three or more covalently linkedoptionally substituted monosaccharides or an amino acid;

R⁵ and R⁶ are independently —H, —CH₃, —CH₂OH, —CHO, —CH₂F, —CH₂Cl,—CH₂Br, —CH₂I;

R⁷ is —CH₂—, —CHF—, —CHCl—, —CHBr—, —CHI—, —C(CH₂)— or —CH(C₁₋₈ alkyl,e.g., —CH(CH₃)—, —CH(C₂H₅)— or —CH(C₃H₇)—);

R⁸ is —CH₂—, —CHF—, —CHCl—, —CHBr—, —CHI—, —C(CH₂)—, —CH(CH₃)—,—CH(C₂H₅)— or —CH(C₃H₇)—;

R⁹ is —CH₂—, —CHOH—, —CHF—, —CHCl—, —CHBr—, —CHI—, —C(CH₂)—, —CH(CH₃)—,—CH(C₂H₅)—, —CH(C₃H₇)—, —CH(OCH₃)—, —CH(OC₂H₅)— or —CH(OC₃H₇)—; and

the hydrogen atom at the 5-position, if present, is in the α- orβ-configuration.

16G. The method of embodiment 15G wherein R¹, if monovalent, is in theβ-configuration.

17G. The method of embodiment 15G wherein R¹, if monovalent, is in theα-configuration.

18G. The method of embodiment 15G, 16G or 17G wherein R², if monovalent,is in the β-configuration.

19G. The method of embodiment 15G, 16G or 17G wherein R², if monovalent,is in the α-configuration.

20G. The method of embodiment 15G, 16G, 17G 18G or 19G wherein R³, ifmonovalent, is in the α-configuration.

21G. The method of embodiment 15G, 16G, 17G 18G or 19G wherein R³, ifmonovalent, is in the β-configuration.

22G. The method of embodiment 15G, 16G, 17G, 18G, 19G, 20G or 21Gwherein R⁴, if monovalent, is in the 6-configuration.

23G. The method of embodiment 15G, 16G, 17G, 18G, 19G, 20G or 21Gwherein R⁴, if monovalent, is in the α-configuration.

24G. The method of any of embodiments 15G, 16G, 17G, 18G, 19G, 20G, 21G,22G or 23G wherein R⁵ and R⁶ are both in the 6-configuration.

25G. The method of any of embodiments 1G to 24G wherein R⁷, R⁸ and R⁹independently are —CH₂—, —CHF—, —CHCl—, —CHBr—, —CHI—, —CH(C₁₋₈ alkyl)-or —CHOH—.

26G. The method of any of embodiments 1G to 25G wherein R⁵ and R⁶independently are —CH₃—CH₂F, —CH₂Cl, —CH₂Br, —CH₂I, or —CH₂OH or whereinR⁵ and R⁶ are both —CH₃.

27G. The method of any of embodiments 15G-26G wherein the formula 1compound comprises 0, 1 or 2 double bonds, wherein the double bonds areoptionally at the 1-2, 4-5 or 5-6 positions, or wherein the double bondsare optionally at the 4-5, 5-6 or 16-17 positions, or wherein the doublebonds are optionally at the 1-2, 4-5 or 16-17 positions, or wherein thedouble bonds are optionally at the 1-2, 5-6 or 16-17 positions.

28G. The method of embodiment 27G wherein the formula 1 compound is16α-bromoepiandrosterone, 16α-bromoepiandrosterone hemihydrate,3β,7β,17β-trihydroxyandrost-5-ene, 3β,17β-dihydroxyandrost-5-ene,3β-hydroxyandrost-5-ene-7,17-dione or 16α-fluoroandrost-5-ene-17-one.

In other embodiments, the formula 1 compounds have the structures shownin embodiment 43D wherein hydrogen atoms at the 5 (if present), 8, 9 and14 positions respectively are in the α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α,β.α.α.α, α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β,β.α.β.β, β.β.α.β, β.β.β.α or β.β.β.β configurations, typically α.α.β.αor β.α.β.α., R¹, R² and R³ independently are a moiety as defined hereinsuch as —H, —OH, ═O, an ester, an ether, a lipid moiety or—O—S(O)(O)—N(R³⁵)₂, where each R³⁵ independently is —H, optionallysubstituted alkyl (e.g., C1-8 alkyl, methyl, ethyl, propyl, butyl orhexyl optionally substituted with one or more —OH, ═O or —O—),optionally substituted alkenyl, a heterocycle, phenyl or benzyl, R⁴ is amoiety as defined herein such as —H, —OH, ═O, an ester, an ether,—C(O)—CH₂OH, —C(O)—CH₂—COOH, —C(O)—CH(O), —C(O)—CH₃, —C(O)—CH₂(halogen),—C(O)—CH₂F, —C(O)—CHF₂, —C(O)—CF₃, —C(O)—CH₂Cl, —C(O)—CHCl₂, —C(O)—CCl₃,—C(O)—CH₂CH₂OH, —C(O)—CH₂CH₃, —C(O)—CH₂CH₂F, —C(O)—CH₂CHF₂,—C(O)—CH₂CF₃, —C(O)—CH₂CH₂Cl, —C(O)—CH₂CHCl₂, —C(O)—CH₂CCl₃,—C(O)—O—CH₂OH, —C(O)—O—CH₃, —C(O)—O—CH₂(halogen), —C(O)—O—CH₂F,—C(O)—O—CHF₂, —C(O)—O—CF₃, —C(O)—O—CH₂Cl, —C(O)—CHCl₂, —C(O)—O—CCl₃,—C(O)—O—CH₂CH₂OH, —C(O)—O—CH₂CH₃, —C(O)—O—CH₂CH₂F, —C(O)—O—CH₂CHF₂,—C(O)—O—CH₂CF₃, —C(O)—O—CH₂CH₂Cl, —C(O)—O—CH₂CHCl₂, —C(O)—O—CH₂CCl₃,—CH(OH)—CH₂OH, —CH(OH)—CH(O), —CH(OH)—CH₃, —CH(OH)—CH₂(halogen),—CH(OH)—CH₂F, —CH(OH)—CHF₂, —CH(OH)—CF₃, —CH(OH)—CH₂Cl, —CH(OH)—CHCl₂,—CH(OH)—CCl₃, —CH(OH)—CH₂CH₂OH, —CH(OH)—CH₂CH₃, —CH(OH)—CH₂CH₂F,—CH(OH)—CH₂CHF₂, —CH(OH)—CH₂CF₃, —CH(OH)—CH₂CH₂Cl, —C(O)—CH₂CHCl₂,—CH(OH)—CH₂CCl₃, —CH(OH)—O—CH₂OH, —CH(OH)—O—CH₃, —CH(OH)—O—CH₂(halogen),—CH(OH)—O—CH₂F, —CH(OH)—O—CHF₂, —CH(OH)—O—CF₃, —CH(OH)—O—CH₂Cl,—CH(OH)—CHCl₂, —CH(OH)—O—CCl₃, —CH(OH)—O—CH₂CH₂OH, —CH(OH)—O—CH₂CH₃,—CH(OH)—O—CH₂CH₂F, —CH(OH)—O—CH₂CHF₂, —CH(OH)—O—CH₂CF₃,—CH(OH)—O—CH₂CH₂Cl, —C(O)—O—CH₂CHCl₂, —CH(OH)—O—CH₂CCl₃ or—CH(CH₃)CH₂CH₂CH(C₂H₅)—CH(CH₃)₃, R⁷ and R⁹ independently are a moiety asdefined herein such as —CH₂—, —O—, —CHOH— with the —OH moiety in the αor β configuration or —C(O)—O and R⁸ is a moiety as defined herein suchas —CH₂—, —O—, —CHOH— with the —OH moiety in the α or β configuration,—C(O)—O, —CH(hydroxyl ester, e.g., a C1-8 ester)-, —CH(alkyl, e.g., aC1-8 alkyl)-, —CH(alkenyl, e.g., C1-8)-, —CH(ether, e.g., C1-8) or—C(OH)₂—. Related formula 1 compounds are optionally substituted at the3, 7 or 17 positions with a second R¹, R² or R⁴ that is not hydrogen,e.g., —CCH, halogen, —OH, alkyl such as methyl, ethyl or butyl oralkynyl such as ethynyl, 1-propynyl or 1-butynyl. For these compounds,double bonds may be absent, or they may be present at, e.g., the 1-2 and4-5 positions or at a single position such as the 4-5 or the 5-6positions. For some of these compounds, R⁵ and R⁶ independently are —H,—CH₃, —CH₂OH, —CH(O) or —C₂H₅. Also, 1, 2 or 3 of R¹⁰ at the 1, 4 or 6positions may independently be a moiety other than hydrogen, e.g., alkylsuch as methyl, a halogen ═CH₂, —CN, —OH or an ester. All of theseformula 1 compounds are suitable for the uses and compositions disclosedherein.

In some embodiments, 1, 2 or more of, e.g., R¹, R², R³, R⁴ and R¹⁰ cancomprise a lipid moiety such as a fatty acid, a monoacylglyceride, adiacylglyceride, a phospholipid, a glycolipid, a sphingolipid or aglycerophospholipid that is esterified, linked through an ether (—O—) oracyl moiety or otherwise bonded to the formula 1 compound. Exemplaryfatty acid esters include —C(O)—(CH₂)_(m)—H where m is 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 15, 17, 19 or 21 and —C(O)—(CH₂), —CH═CH—(CH₂)_(n)—Hwhere each n independently is 1, 2, 3, 4, 5, 6, 7 or 8. Other lipidmoieties that can be bonded to the steroid include phosphatidic acid,phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine andphosphatidylglycerol. The lipid moiety may be bonded to the steroidthrough a hydroxyl or oxygen, phosphate, sulfate or amine at R¹, R², R³,R⁴ or R¹⁰. Such lipid moieties may be bonded to any of the formula 1compounds or genera of formula 1 compounds disclosed herein.

Variations and modifications of these embodiments, the claims and theremaining portions of this disclosure will be apparent to the skilledartisan after a reading thereof. Such variations and modifications arewithin the scope and spirit of this invention. All citations herein areincorporated herein by reference in their entirety. All citations hereinare incorporated herein by reference with specificity.

EXAMPLES

The following examples further illustrate the invention and they are notintended to limit it in any way.

Example 1

16α-bromoepiandrosterone (BrEA) formulation. Two lots of a non-aqueousBrEA formulation were made at a BrEA concentration of 50 mg/mL in 25%polyethylene glycol 300, 12.5% dehydrated ethyl alcohol, 5% benzylbenzoate, and 57.5% propylene glycol as follows. BrEA was obtained fromProcyte, Inc. The remaining excipients are shown below.

Supplier Final Product Excipient Specification Lot No. ConcentrationPropylene glycol USP Arco Chemical 57.5% (v:v) HOC-61220-01104Polyethylene glycol NF Union Carbide   25% (v:v) 300 695752 Dehydratedalcohol USP McCormick 12.5% (v:v) Distilling 97K10 Benzyl benzoate USPSpectrum   5% (v:v) Pharmaceuticals MG025

The formulation was prepared by suspending BrEA in polyethylene glycol300, and sequentially adding propylene glycol, benzyl benzoate, anddehydrated ethyl alcohol to form a solution, which was diluted to thefinal desired volume with additional propylene glycol. The procedure isdescribed below.

The calculated amount of polyethylene glycol 300 was added to acompounding vessel. Then, while mixing, the calculated amount of BrEAwas added to the vessel, and mixed for at least 5 minutes to form asmooth, creamy liquid propylene glycol was added to the vessel, andmixed for a minimum of 5 minutes to form a uniform suspension. Thecalculated amount of benzyl benzoate is added to the vessel, and mixedfor approximately 5 minutes to form a translucent liquid suspension.Dehydrated alcohol was added to the vessel, and mixed for approximately5 minutes to form a clear, colorless solution. Propylene glycol was thenadded to achieve the desired final formulation, and mixed forapproximately 5 minutes. The drug solution was transferred to a volumedispensing device set to deliver 1.2 mL per vial. Under nitrogenpressure, the solution was filtered through two 0.2 μm polyvinylidenefluoride filters in series into 2 cc amber glass vials. The vials werecapped with Teflon-coated, butyl-rubber stoppers and crimp sealed.Materials used in the product vials are listed below.

Material Source Product Code Description Vial Wheaton 2702-B51BA Tubingvial, 2 mL/13 mm, glass, type 1 amber Stopper Omniflex V9239 13 mm,Teflon coated, butyl FM257/2 rubber stopper Seal West 4107 Flip seal, 13mm, mist gray bridge

Product specifications were examined by one or more of the followingassays.

Test Specification Method Physical Examination Clear colorless solutionwith slight alcoholic odor Volume recovery NLT* 1.0 mL USP23<1> Specificgravity TBD USP23<841> Assay for active component 90-110% of label HPLCSterility sterile USP23<71> Endotoxin <0.1 EU/mg USP23<85> Particulatematter ≧10 μm NMT** 6000/cnt USP23<788> ≧25 μm NMT 600/cnt *NLT—no lessthan **NMT—no more than

Lot Analysis Test Specification Lot 1 Lot 2 Physical Clear colorlesssolution Positive Positive Examination with slight alcoholic odor Volumerecovery NLT 1.0 mL 1.15 mL — Specific gravity TBD  1.0411 — Assay foractive 90-110% of label 103.10% 104.25% component Sterility sterilesterile — Endotoxin <0.1 EU/mg 0.024 EU/mg — Particulate matter ≧10 μmNMT 6000/cnt 26 — ≧25 μm NMT 600/cnt 15

Example 2

BrEA drug substance and BrEA formulation stability. An acceleratedstability study of 6 months duration is conducted using BrEA and theformulations from example 1. Samples are taken at 1, 2, 3, 4, 5, and 6month time points and compared with the specifications listed inexample 1. Real time stability (25° C., 60% relative humidity) isconducted using BrEA formulation Lots 1 and 2, with sampling time pointsat 3, 6, 9, 12, 18, 24, and 36 months. After 3 months of storage at 40°C. and 75% relative humidity, the assay potency of BrEA is at least 95%of the label value. The results from the stability testing indicate thatBrEA is stable for at least 3 months at elevated temperature andhumidity in the Lot 1 and 2 formulations.

Example 3

Primate intermittent dosing protocol. Pig-tail Macaque monkeys infectedwith the SHIV₂₂₉ retrovirus were treated with a BrEA formulation asdescribed in example 1. SHIV₂₂₉ is a recombinant retrovirus containingHIV and SIV sequences. J. Thompson et al., abstract #75, 16^(th) AnnualSymposium on Nonhuman Primate Models for AIDS, Oct. 7-10, 1998, Atlanta,Ga., M. Agy et al., abstract #67, 16^(th) Annual Symposium on NonhumanPrimate Models for AIDS, Oct. 7-10, 1998, Atlanta, Ga. In monkeys, itestablishes an aggressive infection that leads to severe symptoms ofend-stage disease in infected untreated animals at about 180-210 daysafter infection. Four pig-tail macaques (2/group) received subcutaneousinjections of the formulation at 1 or 2 mg/kg body weight for 10consecutive days (Protocol 1). On week 8, 3 of the 4 monkeys wereretreated and 2 treatment naïve monkeys were treated with 5 mg/kg of theformulation on an every other day basis for a period of 20 days(Protocol 2). On week 19, all primates receiving treatment began a 3course treatment regimen with 3 mg/kg of the BrEA formulation once dailyfor 10 consecutive days, repeated every four weeks for a total of 3treatment courses (Protocol 3).

The animals were infected with 1-100 TCID₅₀ units administeredintravenously or intrarectally. Viral titers in the first group ofanimals ranged from 10⁶ to 10⁸ before dosing began. All animalsdemonstrated an initial rise in plasma viral SHIV RNA. After a period of2 to 3 weeks, titers began to decline and 3 of the 4 animals showed aresponse to therapy with average viral titers of 0.76 log below baselineat weeks 4 to 5 after initiation of treatment. By week 8, titers in allanimals had returned to baseline values. Blood glucose levels droppedsignificantly, alkaline phosphatase levels were elevated and SGOT/GGTvalues trended towards the high end of normal. No other significantchanges were observed in any of the parameters monitored. The CD4 levelsin all monkeys remained less than 100 cells/mm³ at the end of the firstprotocol.

Three of the five monkeys on the second regimen (Protocol 2) respondedto the BrEA therapy with a greater depth and duration of response thanobserved at the lower dose levels. In the responding animals, theaverage decline below baseline was 1.47 log. The non-responding animalfrom Protocol 1 responded when administered the BrEA formulation inProtocol 2. Two animals did not respond, one each from the treatmentexperienced and treatment naïve groups.

The monkeys on this study were salvaged from an infectivity study andthe first cohort of four monkeys on study (Protocol 1) were expected tolive only a few weeks past the initiation of these experiments as theywere beginning to deteriorate due to disease related causes. One animaldied at day 356 from a toxic reaction to the anesthetic used duringacquisition of a blood sample for analysis. Survival was greater than380 days from the time of infection. Treatment by intermittent dosing ofthe BrEA formulation was used. Three control monkeys were infected with1-10,000 SHIV₂₂₉ TCID₅₀ units and did not receive treatment. Theseanimals are considered the no treatment arm of a survival study. Themean time to death for pig-tailed macaques infected with SHIV₂₂₉ was 193days. Monkeys receiving therapy remained in good clinical health forover 350 days with CD4 levels less than 20 cells/mm³ and withoutopportunistic infections or disease-related symptoms, other than a mildanemia in one animal.

These results show completely unexpected therapeutic responses by theprimates infected with the SHIV retrovirus, which is quite virulent. Theresults show that the majority of subjects in these treatment protocolsnot only had significantly prolonged survival compared to untreatedcontrols, but also the clinical symptoms associated with retroviralinfection improved dramatically, despite the fact that CD4 countsremained low, i.e., less than about 100 CD4 cells/mm³ initially and lessthan about 20 CD4 cells/mm³ later in the treatment protocols. To date,results such as this, i.e., (1) good clinical health in a majority ofsubjects having low CD4 levels (less than about 150 cells/mm³,especially less than about 75 cells/mm³) and (2) no clinical sign ofviral resistance to treatment despite intermittent dosing over aprolonged time period, are unprecedented in primates, humans or anyother animal. The SHIV₂₂₉ model is extremely pathogenic in pig-tailedmacaques. Events that occur in this model over several weeks wouldtypically take several years in humans infected with HIV. Treatment ofmonkeys infected with this virus and treated with commonly usedantiretrovirals, e.g., AZT, 3TC or a protease inhibitor, are notexpected to significantly affect the course of disease progression. Theclinical condition of the animals continues to improve, e.g., weightgain is about 8-15% per animal. These results show that the treatmentusing the intermittent dosing protocol is highly effective despite theapparent impairment of the subject's specific immunity, as shown by thelow CD4 counts. Increased CD4 counts may be attained using immunestimulators such as IL2 or they may increase spontaneously in somesubjects such as humans, depending on the treatment protocol, theduration of dosing or the subject's initial medical condition. Theantiviral effects shown here appear to function at least in part byenhancing the subject's immune responses, e.g., enhanced immune responseby phagocytic cells (NK cells, monocytes and/or macrophages), and/orenhancing any residual specific immune responses, if any, that thesubject may be able to muster.

Example 4

Human treatment protocol. A dose escalation clinical trial is performedusing a nonaqueous formulation containing BrEA or another formula 1compound(s) that is prepared essentially as described in example 1. Thepatients are treatment naïve or treatment experienced and about 3-10patients are examined at each dose level. The initial dose is 25 mg ofBrEA or another formula 1 compound(s) that is administered parenterally,e.g., s.c. or i.m. The dose is administered once or once or twice perday for 1-12 days, followed by no dosing for at least 7 days (e.g., 7 to90 days). Subsequent doses are administered once or once per day for1-12 days, followed by no dosing for at least 7 days (e.g., 7 to 90days). Other dose levels tested are 20 mg, 50 mg, 75 mg, 100 mg, 150 mg,200 mg, 250 mg and 300 mg with each dose administered once per day as asingle dose or as two, three or more subdivided doses. An efficacydosing trial is performed using the same dosing protocol as the doseescalation trial or it may alternatively comprise dosing once or twiceevery other day for 3-17 days, followed by no dosing for 7-90 days andthen repeating the dosing once or twice every other day for 3-17 days.This protocol is repeated indefinitely (e.g., at least about 3-18months) using the optimal dose(s) obtained from the dose escalationtrial, e.g., about 10-200 mg/day of a formula 1 compound.

Example 5

Animal pharmacological studies. Nonclinical studies were conducted usingan oral and a subcutaneous formulation of BrEA. Rats were orallyadministered ¹⁴C BrEA solubilized in different excipients to determinethe levels of drug in blood and various tissues. The results of thesepreliminary pharmacokinetics studies indicated that the absorption ofBrEA by oral administration is about 0.1 to 15%, with at least about 80%excreted in the feces.

The nonaqueous BrEA formulation of example 1 was administered as asingle subcutaneous dose to rabbits. More than 90% of the drug left theinjection site within 24 hours of administration, and reached a maximumconcentration in the plasma of about 1.2% of the injected dose at eightto twelve hours post administration. The circulating half-life of thedrug in the plasma was about twelve hours. The drug did not accumulateto a significant extent in any major organ and was primarily excreted inthe urine.

BrEA was administered subcutaneously to rats using the formulation ofexample 1. Approximately 90% of the drug left the injection site within24 hours of administration, and reached a maximum concentration in theplasma of about 0.2% of the injected dose at one hour postadministration. Elimination from the plasma was biphasic, withhalf-lives of about 12 and 72 hours respectively. BrEA did notaccumulate to a significant extent in any major organ, and was excretedprimarily in the feces. A study is also performed in Rhesus Monkeys withthe example 1 formulation to determine plasma pharmacokinetics.

A pharmacokinetic analysis of ¹⁴C BrEA in plasma was conducted in twofemale Rhesus Monkeys. Trace labeled compound(16α-bromo-3-beta-hydroxy-5α-[4-¹⁴C]-androstan-17-one [50 mCi/mmole])was used at a dose of 1 mg/kg as a subcutaneous injection in thescapular region using an injection volume of 1 mL/kg. The BrEA wasformulated in 25% polyethylene glycol 300, 12.5% absolute ethanol, 5%benzyl benzoate, and qs with propylene glycol. 40 μCi were injected peranimal. Blood samples were taken at 0, 0.5, 1, 2, 4, 8, and 24 hours fordetermination of ¹⁴C activity. The radioactivity in the plasma rose tonear peak concentration in 8 hours and remained at approximately thesame level through the end of the study at 24 hours.

A pharmacokinetic analysis of ¹⁴C BrEA was conducted in New ZealandWhite rabbits. Twenty μCi of ¹⁴C16α-bromo-3-beta-hydroxy-5α-[4-¹⁴C]-androstan-17-one (50 mCi/mmole) plus1 mg/kg unlabeled BrEA was administered to each of three New ZealandWhite rabbits as a subcutaneous injection in the scapular region usingan injection volume of 1 mL/kg. The drug was formulated in 25%polyethylene glycol 300, 12.5% absolute ethanol, 5% benzyl benzoate, andqs with propylene glycol. Blood samples were taken at 0.5, 1, 2, 4, 8,12, 24 hours for all three animals, and at 48 hours for two of theanimals. Twenty-four and 48 hours after administration, one and twoanimals respectively, were sacrificed, and the following organs/tissueswere collected: brain, heart, kidneys, liver, lungs, skeletal muscle,spleen, and injection site muscle and skin. In addition to the organsand tissues, urine and feces were collected as well as the cage wash.BrEA did not accumulate to a significant degree in any of the organslisted above. Of the organs, the greatest mass of drug was observed inthe liver, containing approximately 0.8% and 0.12% of the injected doseat 24 and 48 hours, respectively (average 0.13%).

Percentage of Drug in Organs (Rabbits) Animal 201 Animal 301 Animal 302Organ or Tissue 24 hours 48 hours 48 hours Brain 0.005 0.002 0 Heart0.008 0.003 0.002 Kidneys 0.155 0.055 0.050 Liver 0.76  0.145 0.125Lungs 0.029 0.019 0.011 Spleen 0.002 0 0 Skeletal muscle 0.002 0 0(sample wt. in grams) (3.8 g) (6 g) (5 g) Skin 0.008 0.002 0.004 (samplewt. in grams)   (8 g) (6 g) (9 g)

The average percentages of the administered dose in whole blood wascalculated by multiplying the concentration of drug in whole blood bythe assumed volume of blood in the animals, 200 mL. The amount of drugin the blood reaches a maximum at around 8 hours, and a small amount wasstill evident at 48 hours. The amount of BrEA in whole blood wasconsistently lower than in plasma, suggesting the drug is not taken upto an appreciable extent by red blood cells.

In vivo experiments were conducted to determine the bioavailability ofBrEA via oral administration using different formulations. BrEA was (1)solubilized in soya oil, vitamin E oil, a mixture of vitamin E andcremophore or (2) BrEA was micronized and combined with or without asurfactant. These formulations are described below. The formulationswere administered orally to rats and BrEA levels were determined in theblood, liver, spleen, kidney, and the lymph nodes. In the studies usingmicronized BrEA, the brain was evaluated for drug uptake. Twenty-fourhour urine and feces were collected when BrEA was solubilized in vitaminE and soya oils and vitamin E mixed with Cremophore. The data from thesestudies indicate that BrEA enters into the lymphatics but is eliminatedrapidly from the other tissues. The amount of ¹⁴C radioactivityrecovered in the feces 24 hours after administration was 78 to 83%. Abrief summary of each experiment is provided below and the results areprovided in Table 6.

BrEA (5 mg in 1.0 mL of soya oil or vitamin E oil) supplemented with¹⁴C-labelled BrEA was administered intragastrically to rats.Solubilization of BrEA in the vitamin E or soya oil was facilitated with50 μL ethanol. Animals (3/time point) were assayed at 1.5, 3, 5.5, and24 hours after administration and the ¹⁴C-radioactivity was measured inthe blood, liver, spleen, kidney, lymph nodes and 24 hour feces andurine. The results indicate that, on the basis of ¹⁴C-radioactivity,some of the BrEA is taken into the lymphatic system. The uptake isgreater with soya oil than vitamin E oil in the blood, liver, and lymphnodes.

BrEA (5 mg in 1.0 mL of a vitamin E and cremophore) supplemented with¹⁴C-labelled BrEA was administered intragastrically to rats.Solubilization of BrEA in the vitamin E-cremophore mixture wasfacilitated by the adding 60 μL ethanol. Animals (4/time point) weresacrificed at 2, 3, 5.5, and 24 hours and ¹⁴C-radioactivity was measuredin the blood, liver, spleen, kidney, lymph nodes and 24 hour feces andurine. The results indicate that a small portion of the drug is taken upby the lymphatic system. Judging from the values in plasma, liver andlymph nodes, it appears that drug uptake is slower compared with soy oilor vitamin E and its presence in the tissues is more persistent.

Rats, in groups of three males, were orally administered 1.0 mL of 0.9%NaCl containing 10 or 32 mg BrEA micronized with a surfactant,Synperonic PE/F 127 (2.5% wt/wt). Rats were examined at 1.5, 5 and 24hours after administration. Blood, liver, spleen, kidney, lymph nodes,and brain were assayed for ¹⁴C radioactivity. The levels of BrEA in theblood, in comparison to the experiments with BrEA in Vitamin E oil andsoya, were higher, 0.3% at 1.5 hours, and increased after 5 hours to0.8% and 0.9% of the 10 and 32 mg dose, respectively. Additionally, thevalues in the lymph nodes were similar to those measured at 1.5 hoursand the levels were sustained at 5 hours (5.3 and 5.0%) and 24 hours(3.7 and 3.1%) for the 10 and 32 mg dose, respectively (refer to Table6).

In a repeated dose experiment, rats were intragastrically administered1.0 mL 0.9% NaCl containing 2 mg BrEA micronized with Synperonic PE/F127 (2.5% wt/wt) every 6 to 16 hours. Rats (3/time point) weresacrificed at 40, 72, 84, 90 and 96 hours after the firstadministration. Blood, liver, spleen, kidney and lymph nodes wereassayed for ¹⁴C radioactivity. Higher levels in the blood, liver,kidneys and lymph nodes were noted in this experiment over previousstudies.

Rats, in groups of three males, were orally administered 1.0 mL of 0.9%NaCl containing 2, 4 or 10 mg BrEA micronized without a surfactant. Ratswere sacrificed at 1.5, 5 and 24 hours after administration and blood,liver, spleen, kidney, lymph nodes and brain were assayed for ¹⁴Cradioactivity. The concentration of BrEA micronized without a surfactantin the observed tissues was lower than BrEA plus a surfactant.

Example 6

Inhibition of parasites in vitro. For in vitro antimalarial testing,micro-titer plates were used. The concentration of drugs were preparedas pMol/well according to WHO standard procedures (WHO, 1990). The testcompound was dissolved in 15% DMSO in sterile RPMI-1640. Bothchloroquine sensitive (e.g., WS/97) and resistant (e.g., MN/97) isolatesof Plasmodium species are used.

A schizont inhibition assay was performed as follows. The micro-titerplates were predosed with various concentrations of the test compound.50 μL of parasitised erythrocyte suspension in RPMI-1640 (0.2 mLerythrocyte+0.3 mL serum+4-5 ml RPMI-1640) were dispensed in microtiterwells that contained various concentrations of drug. Triplicate readingswere made for each concentration.

A ³H-hypoxanthine incorporation assay was performed as follows. Thetesting was carried out according to the procedure of Desjardins et al.1979. After 30 hr culture at 37 degrees C., the same microtiter platesfrom schizont inhibition assays with another triplicate wells werepulsed with ³H-hypoxanthine for overnight. The cell suspensions werewashed twice on Millipore glass fiber filter with Millipore filterapparatus. The filter discs were counted for DPM by a Beckman LS6000β-scintillation counter. The activity of the drug was measured byplotting DPM against concentration of drug.

Activity of compounds against Chloroquine sensitive T996/86 P.falciparum in vitro Etianic Etienic Acid Acid Concentration MethylMethyl (μM) DHEA* BrEA* Ester* Ester* 30 65.6 98 60 61.5 15 44 60.1 45.747.4 7.5 38.3 50 40.9 45.3 3.25 37.2 43.7 46 41.4 1.875 23.2 40.9 4143.4 0.938 37.2 31.8 43.3 47.1 IC₅₀ 19.0 μM 7.5 μM 19.5 μM 17.5 μMConcentration (nM) % Inhibition Chloroquine Chloroquine 200 95.9 10094.6 50 97.3 25 94.5 12.5 86.8 6.25 27.2 IC₅₀ 9.0 nM *% inhibition

The activity of 16α-chloroepiandrosterone and16α-bromodehydro-epiandrosterone against chloroquine sensitive T996.86and chloroquine resistant KI P. falciparum in vitro is shown below.

T996.86 KI 16-chloroepiandrosterone IC₅₀ −9.25 pg/mL ~9.25 μg/mL DHEA-BrIC₅₀ −25.0 pg/mL ~25.0 μg/mL

Other formula 1 compounds, e.g., any compound in compound group 1through 25-6 are used in a similar manner to inhibit Plasmodiumparasites.

Example 7

Four-day in vivo protocol for inhibition of Plasmodium berghei. The4-day suppressive test has been widely used and it can be performedwithin a 1 week period. The test consists of the inoculation ofparasitised erythrocytes on the first day of the experiment (D₀),followed by an injection of the test compound, which is alsoadministered on the 2^(nd), 3^(rd) and 4^(th) days of the protocol. Onthe 5^(th) day, blood films are taken and antimalarial activity isassessed either by calculating parasitemia, or by scoring parasitenumbers on a predetermined scale (i.e., 1-5). Peters (Ann. Trop. Med.Parasitol. 64: 25-40, 1970) described a basic procedure using this 4-daytest.

The protocol is summarized as follows. Five female TO mice were used pertest group. P. berghei HP15 ANKA parasites were collected by cardiacpuncture using a heparinised syringe from a donor mouse having a 30+%parasitaemia. The blood was diluted with diluting agent (50% HIFCS+50%sterile PBS) to a final concentration of 1% parasitaemia or 1×10⁷infected erythrocytes per 0.2 mL of the infecting suspension. Each mousewas inoculated intravenously, which produced a more uniform infectionrate than intraperitoneal administration of 0.2 mL of the infectingsuspension. Test compounds were prepared at doses of 100 mg/kg in (16.7%DMSO+83.3% Celacol). The steroid formulations were administeredintraperitoneally 2 hours after parasite inoculation. The compounds wereadministered once a day starting on D₀, and continued on the followingthree days. Blood films were made from tail blood on the day after thelast dosing of compound and the blood was fixed with 100% methanol andstained with 10% Giemsa. Parasitaemias were scored on a scale of 0-5,where 5 is equal to the control.

An inoculum of 1% parasitaemia 1×10⁷ erythrocytes/mL, 0.2 mL per mouse(female strain TO mice), was delivered by intravenous injection. Drugadministration commenced 2 hours after inoculation on Day 1 andcontinued for 3 days. The results are shown below from blood films fromall 20 mice on Day 5 when parasitaemias were assessed.

Compound Treatment Parasitaemia Score (0-5) BrEA 100 mg/kg × 4 daysi.p.* 1 Etienic Acid 100 mg/kg × 4 days i.p. 2 DHEA 100 mg/kg × 4 daysi.p. 1 Chloroquine  3 mg/kg × 4 days i.p. 1 control N/A 5 *i.p. =intraperitoneal injection

In a similar protocol, mice are inoculated with a solution containing1×10⁷ erythrocytes/mL by I.V. injection. Two hours later give drug isdelivered by I.V. injection. BrEA or another formula 1 compound is given(0.2 mL I.V. or S.C.) once a day for 4 days. Tail snips are used toobtain blood after the study. Mice infected with P. berghei were used toobtain infected cells. Parasites are harvested from cardiac mouse blood,and uninfected mice are infected using 0.2 ml of blood with 14%parasitaemia per mouse I.V. Two hours later, the first dose of BrEA (100mg/kg I.V. or S.C.) is delivered to the infected animals. The BrEAformulation was a sterile solution containing 15 mg/mL of BrEA in 45%hydroxypropyl-β-cyclodextrin and 0.9% saline. At 1, 2, 3 and 4 daysafter the infection of the animals, BrEA (100 mg/kg I.V. or S.C.) isdelivered to the infected animals. No deaths occurred in the groupreceiving I.V. BrEA at day 30, but all control animals were dead by day10. All animals treated with BrEA by S.C. delivery were dead by Day 11.

Example 8

Rat in vitro and in vivo study. In the in vitro protocol the parasite(Plasmodium falciparum, chloroquine sensitive strain WT and chloroquineresistant strain Dd2) level is adjusted to 1% and the hemocrit isadjusted to 7% with medium. Using a 96 well plate, 50 μL of parasite and100 μL of drug mixed with media are added to each well and the procedureis done in triplicate. The plate is placed in a chamber containing aphysiological gas mixture and incubated at 37° C. The media/drug mixtureis changed at 24, 48 and 72 hours. On day 5 (96 hours) slides of eachwell are made, stained with Gemsia and 500 red blood cells are countedfor each slide. The triplicates are averaged and data are reported inpercent inhibition.

In the in vivo protocol, Lewis rats weighing 80-85 grams were given astandardized IP injection of parasite (Plasmodium berghei). Rats werethen intravenously injected 2 hours later with one of the treatmentsdescribed in the table below, returned to their housing, fed standardlab chow and allowed free access to water. Animals were weighed andtreated again 24, 48, and 72 hours after the first treatment and againreturned to their housing and they were allowed free access to food andwater. The animals were weighed again and then bled using a 26-gaugeneedle on day 5, 11 and 28 post inoculation. Hemocrits were measured andblood smears are prepared for each rat. The blood smears were thenstained using Gemsia and the level of parasitemia (defined as thepercent of red cells with parasites) were determined. Animals were againreturned to their housing and observed twice daily for evidence ofprogressive disease, defined as listlessness and or adverse drugreaction, which is defined as a loss of 20% of original body weight, fora total of 28 days. If either progressive disease or drug reaction isnoted, the animals are euthanized.

The BrEA formulation was a sterile solution containing 15 mg/mL of BrEAin 45% hydroxypropyl-β-cyclodextrin and 0.9% saline.

Group 1 Group 2 Group 3 Group 4 Control 0.9% Chloroquine BrEA Low DoseBrEA saline Control 30 mg/kg High 40 mg/kg Dose 60 mg/kg

The intravenous injections were given on days 0, 1, 2 and 3 and theresults are shown below. The results showed that treatment in vivo witha formulation comprising BrEA reduced parasitemia to a level comparableto that seen with the chloroquine (“Clq”) control. The results aresummarized in the table below.

% RBC parasitemia Day 4 saline control 16% chloroquine control 10% lowdose BrEA 9% high dose BrEA 7% Day 11 saline control 36% chloroquinecontrol 16% low dose BrEA 12% high dose BrEA 11%

Example 9

Human clinical study—parasite infection. Response to drug treatment wasgraded as per World Health Organization criteria (WHO 1973) in infectedpatients. Evaluation of therapeutic response was determined using theparasitic and fever clearance times. Parasite clearance was expressed asthree indices; the time for the parasite count to fall by 50% of thepre-treatment (baseline) value (PC₅₀), (ii) the time for the parasitecount to fall by 90% of the baseline value (PC₉₀) and (iii) the time forthe parasite count to fall below the level of microscopic detection(parasite clearance time PCT) (N. J. White and S. Krishna Trans. R. Soc.Trop. Med. Hyg. 83: 767-777, 1989; White et al., J. Infect. Dis. 165:599-600, 1992; White et al., J. Infect. Dis. 166: 1195-1196, 1992). Thefever clearance time was defined as the time from drug administrationtill the oral or rectal temperature fell to or below 37.2° C. andremained so for at least 48 h.

Venous blood (5 mL) was obtained from two patients before treatment andat 4, 6, 8, 12, 18, 20, 24, 30 and 36 h after treatment or at 4 or6-hourly intervals after treatment until there was complete clearance ofperipheral parasitemia. Blood was collected aseptically and transferredto 10 mL syringes containing 2 mL of acid citrate dextrose (ACD) for invitro culture. Prior to incubation, the plasma was separated from thered blood cells and the red blood cells were washed twice. Parasiteswere cultured by modification of standard in vitro culture techniques(W. Trager and J. B. Jensen, Science 193:673-675, 1976; A. M. Oduola etal., J. Protozool. 39: 605-608, 1992). Samples were dispensed intosterile centrifuge cubes within 10 min of collection and spun down. Thesupernatant plasma was stored while the packed cells were washed twicewith culture medium (washing medium, RPMI-1640 medium, containing 25 mMHEPES buffer and 25 mmol/L NaOH). The buffy coat was removed by vacuumaspiration. A 1:10 fold dilution was done for each blood sample withcomplete washing medium [CMP (washing medium supplemented with 10% humanplasma)]. One milliliter each of the sample was transferred into 2 wellsof a 24 well micro culture plate. Cultures were incubated at 37 degreesC. in an atmosphere of 5% CO₂, 5% 0₂ and 90% N₂ premixed gas. Theculture medium was changed daily and thin blood smears were prepared formicroscopy at 24 and 48 h after the culture has been set up. The culturesamples were diluted with unparasitized washed type A Rh-positive redblood cells if the proportion of parasitized red blood cells was morethan 2%.

Microscopy. During the in vivo study, thin and thick blood films werefixed with dehydrated methanol (100%) and heat, respectively, werestained with 10% Giemsa for 20 min. Parasitemia was quantified in thinfilms by counting 2000 red blood cells in clear contiguous fields andfinding the proportion that was parasitized. In thick films, parasitemiawas quantified by counting parasites against leukocytes. A film wasdeclared negative if no parasites were found after examination of 200microscope fields of a thick smear. During in vitro and ex vivo study,pretreatment thin and thick smears were, graded for ring stages by themethod of Jiang as modified by Li et al. (J. B. Jiang et al, Lancet2(8293): 285-288, 1982; K. Silamut and N. J. White Trans. R. Soc. Trop.Med. Hyg. 87: 436-443, 1993; X. L. Li et al, Chi. J. Parasitol. Dis. 12:296, 1994). Approximately 5000 erythrocytes were counted in clearcontiguous fields 24 and 48 h after incubation of blood obtained at eachtime point and graded for maturity into tiny rings, small rings, largerings, pigmented trophozoites and schizonts. Functional viability wasestimated as the percentage of asexual ring forms capable of maturing topigmented trophozoites or schizonts after 24-48 h of in vitro culture(W. M. Watkins et al., Trans. R. Soc. Trop. Med. Hyg. 87: 75-78, 1993).

Calculation of parameters. The patients presented with acute symptomaticsevere non-cerebral pure P. falciparum malaria. They had oral fluidintolerance, body temperatures greater than 39° C., greater than 5000parasites per micro liter of blood, asexual parasitemia and they had anegative urine test for antimalarial drugs. They were administered 25 mLintravenously every four hours with BrEA suspended in sterile 45%β-cyclodextrin in saline at a concentration of 25 mg/mL. This regimenwas continued for four days. Parasitemia quantification and clinicalexamination were done once every 6 hours for the first 72 hours,followed by daily assessment of the parameters up to day 7 (168 hrs) andthereafter on day 14.

Blood films were Giemsa-stained and parasitemia quantification was donein thick films by counting 2000 parasites against leukocytes, and thethin films by finding the proportion of infected red blood cells.Response to drug treatment was graded according to WHO criteria.Evaluation of therapeutic response was done using the parasitic andfever clearance times. Parasite clearance was expressed as threeindices: The time for the parasite count to fall by 50% of thepre-treatment (baseline) value (PC₆₀); to fall by 90% of the baselinevalue (PC₉₀); and to fall below the level of microscopic detection(parasite clearance time) PCT.

The fever clearance time was defined as the time from drugadministration until the oral/rectal temperature fell to below 37.2degrees C. and remained so for greater than 48 hours. The parasiteclearance rate at day 14 was 100%. The clinical response thus includedan effect on parasitemia in both patients and amelioration of one ormore symptoms of infection.

Intravenous BrEA Malaria Patient Trial Patient A Patient B Feverclearance time 12 hrs 18 hrs Parasite clearance times Time to 50%clearance 18 hrs 24 hrs Time to 90% clearance 24 hrs 48 hrs Time to 100%clearance 48 hrs 64 hrs

Example 10

Cellular studies in vitro. The effect of BrEA on pentosephosphate shunt(PPS) activity in normal human RBC was examined using whole cells. Sinceglucose-6-phosphate dehydrogenase (“G6PD”) is the limiting enzyme of thePPS, PPS flux measurement is considered to better reflect G6PD activityin the whole cell compared to G6PD activity measurement in a celllysate. G6PD activity measured in a cell lysate is typically about1100-fold higher than the PPS flux in whole resting unstimulated RBC(G6PD activity in cell lysate: 165; PPS flux 0.142 micromoles/hour/mlRBC). PPS flux and G6PD activity in the whole RBC depends on a number offactors (the concentration of NADPH, NAD, and ATP, and intracellularpH), which are kept constant if the measurement is performed in thelysate and may vary in the whole RBC. Levels of G6PD activity in cellsis considerably above normal basal needs and inhibition of overall G6PDactivity might have no or minor consequence on PPS flux in the wholecell. For example, RBC with the Mediterranean G6PD mutant with about 1-3percent residual activity compared with normal individuals have noimpairment in basal PPS flux, but show impaired flux when flux throughPPS is stimulated by methylene blue addition. A series of experimentswere performed using varying amounts of BrEA and PPS flux was measuredin unstimulated basal RBC and in methylene-blue (MB)-stimulated RBC.

The data below shows PPS flux (micromoles/hour/ml RBC) in basalunstimulated, and MB-stimulated normal RBC. Different concentrations ofBrEA (0.3, 3.5 and 7 micromolar, final) were supplemented to suspensionsof washed RBC suspended in RPMI, pH 7.4 at 10% hematocrit, whereby PPSflux was immediately measured without further incubation and withoutfurther washings. A minor inhibition of MB-stimulated PPS flux wasobserved with BrEA at 7 μM.

PPS flux control, unstimulated RBC 230 DMSO control, unstimulated RBC270 DMSO control, MB stimulated RBC 5090 0.3 μM BrEA, unstimulated 2500.3 μM BrEA, MB stimulated 5000 3.5 μM BrEA, unstimulated 270 3.5 μMBrEA, MB stimulated 4950   7 μM BrEA, unstimulated 295   7 μM BrEA, MBstimulated 4660

The data below shows average values of 3 experiments, where basal,unstimulated, and MB-stimulated PPS flux (micromoles/hour/ml RBC) wasmeasured in normal RBC. In these experiments, different concentrationsof BrEA ˜0.8, 8 and 80 micromolar, final) were supplemented tosuspensions of washed RBC suspended in RPMI, pH 7.4 at 10% hematocrit.After a 90-min incubation at 37° C. with and without BrEA, PPS flux wasmeasured. The results showed a dose-dependent inhibition ofMB-stimulated PPS flux. Inhibition was 10% at 8 micromolar (p=0.006 vscontrol+DMSO) and 25% at 80 micromolar (p=0.002 vs control+DMSO).

PPS flux control, unstimulated RBC 430 control, MB stimulated RBC 5410DMSO control, unstimulated RBC 480 DMSO control, MB stimulated RBC 48900.8 μM BrEA, unstimulated 410 0.8 μM BrEA, MB stimulated 4930   8 μMBrEA, unstimulated 450   8 μM BrEA, MB stimulated 4430  80 μM BrEA,unstimulated 450  80 μM BrEA, MB stimulated 3660

Example 11

Inhibition of parasite growth. The effect of Epi(16α-bromoepiandrosterone) on parasite (Plasmodium falciparum) growthwas shown. Epi was active at a concentration of 1 μM.

Parasitemia after treatment Time 0 24 hrs 48 hrs 72 hrs control + DMSO5% 5.40% 3.10% 5.20% Epi 1 μM 5% 5.70% 5.50% 1.60% Epi 10 μM 5% 5.60%0.90% 0 Epi 100 μM 5% 0 0 0 Epi 500 μM 5% 0 0 0 control + DMSO 2% 8.80%  11%   8% Epi 50 nM 2% 9.90% 9.20% 8.30% Epi 1 μM 2% 5.80% 6.10% 2.10%Epi 2.5 μM 2% 7.30% 5.80% 3.20% Epi 5 μM 2% 5.40%   6% 1.80% Epi 10 μM2% 4.20%   3% 0 Epi 50 μM 2% 0 0 0

Parasitemias were determined by standard methods (microscopic inspectionof at least 500 cells, stained with Diff-Quick™ (Baxter). Parasites werecultured under standard conditions in RPMI-1640 supplemented withHepes/Glucose (10 mM), glutamine (0.3 g/liter) and 10% human plasma. Thehematocrit was 1%.

Example 12

Stimulation of phagocytosis. The capacity of BrEA to influencephagocytosis of Plasmodium parasite-infected RBC is examined usingadherent human monocytes. The parasitemia level is about 8-10% and humanmonocytes are obtained from buffy coats from blood as follows.Peripheral blood mononuclear cells are separated from freshly collectedplatelet-poor buffy coats discarded from blood samples of healthy adultdonors of both sexes. Separated cells are washed once with luke-warm PBSsupplemented with 10 mM glucose (PBS-G) and resuspended at 5×10⁶cells/mL in ice-cold RPMI 1640 medium supplemented with 23 mM NaHCO₃ and25 mM Hepes, pH 7.4 (RMBH). Dynabeads M450 Pan B and Pan T (Dynal) areadded to cells in a 4:1 ratio for 20 min at 4° C. B-lymphocytes andT-lymphocytes are removed as specified by the manufacturer. Theremaining monocytes are washed 2 times in RMBH, resuspended in AIM Vcell culture medium (Gibco) at 1×10⁶ cell/mL. The monocyte layer iscollected, washed with PBS-G at 37° C. and resuspended in AIM V mediumat 1×10⁶ cells/mL. Purified cells are >90% monocytes as assessed by CD14expression.

Phagocytosis of opsonized parasitized RBC (PE) is determined as follows.Phagocytosis of fresh-serum opsonized PE is initiated by mixing 10PE/monocyte. Suspensions are briefly centrifuged (150×g for 5 sec atroom temperature) to improve contact between PE and monocytes. To avoidattachment of monocytes after centrifugation and during the wholeincubation period, cells are kept in suspension at 5×10⁶ cells/5 mL AIMV medium in 6 cm diameter teflon bottom dishes (Heraeus) in a humidifiedincubator (95% air, 5% CO₂) at 37° C. On average, at least 90% of themonocytes phagocytose PE, as assessed by microscopic inspection. Controlcells are kept under similar conditions without phagocytosis.Quantitative assessment of phagocytosis is performed by a previouslydescribed bioluminescence method (E. Schwarzer, et al., Br. J. Haematol.1994 88: 740-745).

Erythrocyte treatments and parasite cultures are as follows. Fresh blood(Rh+) is used to isolate erythrocytes (RBC). Washed RBC are infectedwith schizont/trophozoite parasite stages (Palo Alto strain,mycoplasma-free). Stage specific parasites are isolated by thePercoll-mannitol method. Briefly, normal schizont-stage parasitizedRBC(SPE) separated on Percoll-mannitol gradient (parasitemia>95% SPE)are mixed with RBC suspended in growth medium (RPMI 1640 mediumcontaining 25 mmol/L Hepes, 20 mmol/L glucose, 2 mmol/L glutamine, 24mmol/L NaHCO₃, 32 mg/L gentamicin and 10% AB or A human serum, pH 7.30)to start synchronous cultures at selected hematocrit values. Theinoculum parasitemia is adjusted to 20% normal SPE for isolation of ringparasitized RBC (RPE) and to 5% normal SPE for isolation oftrophozoite-stage parasitized RBC (TPE). At 14-18 hours after inoculumparasites are at ring-stage in the first cycle; at 34-33 hours,parasites are at trophozoite-stage in the first cycle; and at 40-44hours after inoculum parasites are at schizont-stage in the first cycle.RPE, TPE and SPE are separated on Percoll-mannitol gradients. Theparasitemia is usually 8-10% RPE, and >95% TPE. Nonparasitized andparasitized RBC are counted electronically. To assess total parasitemiaand relative contribution of RPE, TPE and SPE, slides are prepared fromcultures at indicated times, stained with Diff-Quik™ parasite stain andabout 400-1000 cells are examined microscopically.

The effect of a formula 1 compound such as BrEA in parasitized RBC isexamined using various concentrations of the compound, e.g., BrEA, e.g.,0.5 μM, 1 μM, 10 μM, 25 μM and 50 μM. Trophozoite-parasitized RBC,schizont-parasitized RBC or ring-parasitized RBC are examined asdescribed.

Example 13

Human malaria clinical trial. The clinical trial protocol thatincorporates about 15-20 patients is established. For a phase I, I/II orII trial, the patients are mildly infected with one or more Plasmodiumparasites and they are mildly symptomatic (less than about 8-10%parasitemia of RBC). Before treatment, the patients are optionallytested for infection with HIV, HCV, TB, and Cryptosporidium. Patientswith one or more co-infections are given standard care for thecoinfection. The patients are hospitalized for treatment for one week.Two or more dose groups, e.g., 25, 50 or 100 mg/day of BrEA administeredparenterally, e.g., by intramuscular, subcutaneous or intravenousinjection, on 3, 4 or 5 days of the week when patients are dosed. Dosingis on consecutive days or on an intermittent schedule, e.g., 2, 3 or 4doses with one dose administered every other day.

The formulation containing BrEA is as described herein, e.g., theformulation of example 1 or a formulation that comprises 100 mg/mL BrEA,PEG300-30% v/v, propylene glycol 30% v/v, benzyl benzoate 30% v/v andbenzyl alcohol 2% v/v. At day 5-7, if less than about 50% reduction inparasitemia is observed, the patients are given standard care formalaria (mefloquine). During the week of treatment and for 1, 2 3, ormore weeks there after, blood samples are taken periodically forevaluation of parasitemia, pharmacokinetics, plasma cytokines (e.g.,IL-2, IL-4, IL-10, IGF1, γIFN, GM-CSF), and intracellular cytokines(e.g., IL-2, IL-4, IL-10, IGF1, γIFN, GM-CSF). The patients areoptionally treated again at about 2 to 12 weeks after the initialdosing, using the same or a similar protocol as that used in the initialdosing protocol.

An exemplary open-label study of a BrEA formulation administeredintramuscularly to semi-immune patients with uncomplicated malaria isconducted. The formulation comprises 100 mg/mL BrEA, PEG300 ˜30% v/v,propylene glycol 30% v/v, benzyl benzoate 30% v/v and benzyl alcohol 2%.Patients will remain at the hospital as in-patients for the first 7 daysof the study. Patients will receive one daily intramuscularadministration of 50 mg or 100 mg of BrEA for 5 consecutive days. Dailyevaluation for the first 7 days, and up to study day 14, may includeparasitemia evaluation (twice daily), chemistry, hematology and druglevels (pharmacokinetic evaluation). If, after study day 7, theparasitemia levels decrease from the screening value and the patient isclinically stable, the patient may be followed on a daily basis forparasitemia (twice daily) for up to an additional 7 days as hospitalin-patients. If a patient becomes clinically unstable at any time duringthe study, the patient will be discontinued and may be offered thestandard treatment for malaria. Patients deficient inglucose-6-phosphate dehydrogenase enzyme may be excluded, since BrEAinhibits the enzyme. Other considerations that may lead to exclusion ofpatients from the trial include patients diagnosed with any of thefollowing: severe anemia (hematocrit <21% or hemoglobin <7 g/dL); renalor liver failure by history and/or laboratory results respiratorydistress as evidenced by dyspnea or respiratory rate ≧30 per minute;hypotension (systolic blood pressure <90 mm Hg); tachycardia (heartrate >130 beats/minute); pregnant or breast-feeding women; significantactive co-morbid illness (acute medical diagnosis requiring specifictherapy; patients with parasitemia >10% on peripheral smear.

Blood samples may be collected from each patient for future clinicalevaluation such as the determination of activation markers orimmunological analyses (e.g., assay for intracellular or extracellularinterleukins IL-1β, IL-2, IL-4, IL-6, IL-10 and IL-12, γIFN and TNFα).

Example 14

Liposome formulation. Liposomes suitable for parenteral administrationare prepared as follows. 400 mg of phosphatidyl choline and 80 mg ofBrEA are dissolved in chloroform and methanol (2:1 v/v) and the solutionis dried by rotary evaporation under reduced pressure. The resultingfilm is rehydrated by adding 8.0 mL of a 0.9% w/v NaCl solution andagitating the solution. The sizes of the liposomes are optionallymeasured, e.g., by photon correlation spectroscopy (Malvern Zetasizer3000 or equivalent). The liposomes are optionally sized by, e.g.,sonication to reduce the average size below 400 nm, or by filtrationusing suitable filters. Similar procedures are used to prepare liposomepreparations that contain a formula 1 compound at about 15-100 mg/mL.The formulation is used to deliver the compound orally or parenterally(I.M., S.C., I.V.).

Example 15

Cyclodextrin formulation. A cyclodextrin formulation containing BrEA isprepared as follows. 450 g of hydroxypropyl-β-cyclodextrin is added toabout 1 L of ethanol and the mixture is stirred for about 4-24 hours,until a clear solution is obtained. Non-micronized or micronized BrEA isadded to give a concentration of 20 mg/mL and the mixture is stirreduntil a clear solution is obtained. The solution is dried, e.g., byrotary evaporation under reduced pressure. The dried material is addedto 1 L of physiological saline and stirred until a clear solution isobtained. The solution is sterilized by filtration using a 0.2 μm poresize filter and dispensed into sterile containers. Similar proceduresare used to prepare cyclodextrin formulations that contain a formula 1compound at about 10-100 mg/mL. The formulation is used to deliver thecompound orally, parenterally (I.M., S.C., I.V.) or by a buccal orsublingual route.

Example 16

Suppository formulation. A suppository formulation containing a formula1 compound such as BrEA is prepared as follows. Sufficientnon-micronized BrEA is measured to obtain a desired number of units thatcomprise 500 mg each of BrEA. The BrEA is blended with a suppositorybase, e.g., triglyceride from edible vegetable oil, to provide desiredcharacteristics, e.g., a free fatty acid content of about 0.1% w/w, asaponification value of about 242, an iodine value of about 3, moistureat about 0.1% w/w and a closed capillary melting point of about 35° C.

Example 17

Human HCV clinical trial. A female patient infected with HIV and HCV wasdosed I.V. with BrEA for 3 consecutive days using a formulation thatcontained 20 mg/mL BrEA in 45% w/v hydroxypropyl-6-cyclodextrin andsaline. Four mL of the formulation (80 mg BrEA) was administered to thepatient every 4 hours during the 3 day treatment period. The patient'spredosing HCV level was 6.5 Log₁₀) as measured by PCR and the HCV levelwas 6.2 Log₁₀) on the first day of dosing, 5.5 Log₁₀) on the 3^(rd) dayof dosing and 4.9 Log₁₀) three days after the last dose wasadministered. HIV RNA levels as measured by PCR was 5.2 Log₁₀)(predosing), 5.8 Log₁₀) (first day), 5.9 Log₁₀) (third day) and 5.4Log₁₀) (day 6). The NK cell counts (cells/mm³) were 28, 41 and 38 atpredosing, day 0 and day 3.

Example 18

A formulation comprising 100 mg/mL BrEA, ˜30% v/v PEG300, 30% v/vpropylene glycol, 30% v/v benzyl benzoate and 2% v/v benzyl alcohol wasprepared by suspending BrEA in polyethylene glycol 300, and sequentiallyadding propylene glycol and benzyl benzoate, to form a solution, whichwas diluted to the final desired volume with additional propyleneglycol. The procedure is described below.

The calculated amount of polyethylene glycol 300 was added to acompounding vessel. Then, while mixing, the calculated amount of BrEAwas added to the vessel, and mixed for at least 5 minutes to form asmooth, creamy liquid propylene glycol was added to the vessel, andmixed for a minimum of 5 minutes to form a uniform suspension. Thecalculated amount of benzyl benzoate is added to the vessel, and mixedfor approximately 5 minutes to form a translucent liquid suspension.Propylene glycol was then added to achieve the desired finalformulation, and mixed for approximately 5 minutes. The drug solutionwas transferred to a volume dispensing device set to deliver 1.2 mL pervial. Under nitrogen pressure, the solution was filtered through two 0.2μm polyvinylidene fluoride filters in series into 2 cc amber glassvials. The vials were capped with Teflon-coated, butyl-rubber stoppersand crimp sealed.

Example 19

Opportunistic infection clinical protocol. A double blind, randomized,placebo controlled study of 100 mg of BrEA administered intramuscularlyto late stage HIV-infected patients at risk for opportunistic infections(Ols). HIV-1 seropositive patients with a CD4 cell count ≦100 cells/mm³,HIV RNA at 1×10⁶ copies/mL and a Karnofsky score of at least 60 areidentified for potential inclusion into the protocol. Patients in allclinical protocols must understand and sign a written informed consentform prior to screening evaluations.

BrEA in the formulation of example 16 is used. Administration of drug orvehicle will be for 3 to 5 consecutive days followed by about 35-90 daysof observation, e.g., 37 days of observation. An exemplary treatmentregimen comprises 5 days of treatment followed by 37 days ofobservation, which is repeated for a total of 7 courses over 42 weeks.The incidence rate of Ols as well as the time to resolution or controlof the Ols will be monitored and compared to a placebo control group.The patients may be monitored monthly for 2 or 3 months after completionof the study for follow-up. The incidence of Ols or conditionsassociated with AIDS are monitored, e.g., as tuberculosis (TB),candadiasis, Pneumocystis pneumonia (PCP), diarrhea, or Kaposi'ssarcoma, may be evaluated as protocol endpoints. If a patient isdiagnosed with one or more of the protocol specified opportunisticinfections, the protocol regimen a treatment for the OI will beinitiated, e.g., Fluconazole for Candidiasis or for PCP, trimethoprimand sulfamethoxazole or Dapsone. A similar protocol is used with otherformula 1 compounds.

Example 20

Human HIV clinical protocol. Patients infected with HIV are dosed withan i.m. injection of 25-200 mg of BrEA using a formulation containing100 mg/mL BrEA, PEG300 ˜30% v/v, propylene glycol 30% v/v, benzylbenzoate 30% v/v and benzyl alcohol 2% v/v. The pateints are dosed onceper day for 5 consecutive days followed by a period of about 28 days orlonger with no BrEA treatment. The patients were them provided with onemore course of 5 consecutive days of dosing with BrEA, followed by anon-dosing period of at least about 28 days. Up to 8 rounds of 5-daytreatments, followed by at least 28 days of no dosing were provided.Immunological responses were then assayed using blood or plasma samplesfrom the patients by flow cytometry and other known analytical methods.Immune cell subsets or other measured markers were assayed within 24hours of obtaining the sample from each patient. Labeled antibodies,e.g., anti-CD antigen antibodies conjugated with fluorescent dyes (FITC,phycoerythrin, allophycocyanin or PerCP), were prepared and usedessentially according to standard protocols using commercially availablereagents, see, e.g., PharMingen, 1998 Research Products Catalog,technical protocols at pages 732-774, human cell surface molecules atpages 182-295 and mouse, rat and hamster cell surface molecules at pages2-173 and cytokine and chemokine reagents at pages 344-489.

Otherwise untreated patients with CD4 counts of ≧200/4 and HIV-1 viralRNA between 5,000 and 1,000,000 copies/mL (branched-DNA, version 3.0,Bayer, Tarrytown, N.Y.) provided informed consent and were enrolled intoa Phase I/II dose-escalation study of the safety and efficacy ofintramuscular injections of BrEA. The protocol was conducted in SouthAfrica. In the first cohort, four patients received an initial injectionof 1 mL (50 mg), followed by a safety and pharmocokinetic study forseven days, and then received five daily injections of 1 mL (50 mg). Thesecond cohort of 8 patients was randomized in a 1:2 ratio to receiveeither 50 mg (1 mL) or 100 mg (2 mL) on the same schedule. Patients wereallowed at the discretion of the investigators to receive up to threeadditional courses each, repeated after 28 days of observation. Theprotocol was amended to introduce an improved formulation, and to extendthe initial pharmacokinetic and safety period to 14 days, theobservation period following the five daily doses to 35 days, and toallow up to 7 treatment courses. A third cohort of 24 patients wasrandomized in a 1:2:4 ratio to receive either 50 mg (0.5 mL), 100 mg (1mL), or 200 mg (2 mL) of BrEA. The patients were all male, since initialsafety studies are not permitted in females in South Africa. Within thegroup of 39 patients randomized, the ethnic demographic included was 18Caucasian, 17 African, and 4 other. The average age was 34 (range20-63), the average initial CD4/4 was 434 (range 176-1210), and theaverage viral load was 13,772 copies/mL (range 3,020-158,489). Patientsunderwent physical examinations and provided samples for chemistry,hematology, virology and immunology testing approximately every twoweeks.

The clinical protocol is a phase I/II, open-label, randomized study of 3dose levels of BrEA administered intramuscularly to HIV-infectedpatients who are treatment naïve. There will be 3 treatment groups andeach group will consist of 2 parts (Parts A and B). Patients willreceive the same dosage of BrEA throughout Parts A and B of the study.If a patient experiences an antiviral response (an HIV RNA titer atleast 0.5 log below the average of the screening and baseline values) orbenefits (any decrease in HIV RNA titers below the average of thescreening and baseline values) from the treatment received during PartsA and B of the study, the patient may continue receiving 5-day treatmentcourses of the BrEA formulation of example 2 at the dose initiallyreceived. This treatment course may be repeated up to 30 times or more.

All patients may be monitored for levels of HIV RNA (Chiron Quantiplex™branched chain DNA assay), T-cell subsets [CD4/CD8], proviral HIV DNA(PBMC), interleukins [IL-2, 4, 6, 8, 10, and 12] (serum), γIFN (serum),insulin-like growth factor [IGF-1] (serum) and tumor necrosis factor[TNF] (serum) throughout the study. PBMC quantitative co-culture (cells)may be conducted on a subset of patient samples. Assays for additionalactivation markers may be conducted. Analysis of chemistry andhematology panels and urinalysis is planned. Additionally, patientsco-infected with hepatitis B and/or C viruses, malaria or tuberculosismay be monitored regularly for viral titers or microbiological cultures.Serial blood and urine samples will be collected from a subset ofpatients for pharmacokinetic determination after the first dose on PartA and the last dose on Part B.

Treatment may consist of more than one intramuscular injection.Intramuscular injections may be administered in different locations(i.e., left or right upper arms or thighs or buttocks) and a single 100mg or 200 mg dose of BrEA may be delivered to patients in two or moresubdoses of less than 100 mg (e.g., 50 mg).

There are two segments of this study, Segment 1 and 2. Both segmentsconsist of two parts, Part A and Part B. The first 12 patients enrolledon the study will be assigned to the design described in Segment 1. Theremaining 24 patients will be assigned to Segment 2 of the study. Thedesign of each segment is provided below.

Part A will consist of a single intramuscular injection of a BrEAformulation. The day the patient receives the injection will be studyday 1. Patients participating in the pharmacokinetic subgroup will haveserial blood and urine samples collected, beginning on study day 1. PartB of the study begins on study day 8 (Segment 1) or study day 15(Segment 2).

Segment 1 Part B consists of 5 consecutive daily intramuscularinjections of the formulation of example 1 at the same dose as receivedin Part A of the study. The day the patient receives the first dose willbe on about study day 8-12. The 5-day treatment course is followed by anapproximate 28-day observation period (or approximately 32 days from afirst dose on day 8 to the initiation of a second treatment course onday 40). During the observation period, patients will be asked to returnto the clinic on a weekly basis for various tests. Patientsparticipating in the pharmacokinetic subgroup will have serial blood andurine samples collected, beginning approximately on study day 12-17.

Segment 2 Part B consists of 5 consecutive daily intramuscularinjections of the formulation of example 2 at the same dose the patientreceived during Part A of the study. The day the patient receives thefirst dose will be about at study day 15. The 5-day treatment course isfollowed by an approximate 45 day observation period (or approximately49 days from the first dose on study day 15 to the initiation of thenext treatment course on study day 64). During the observation period,patients will be asked to return to the clinic on a weekly basis forvarious tests. Patients participating in the pharmacokinetic subgroupwill have serial blood and urine samples collected, beginningapproximately on study day 19.

Randomization in this dose escalation study is as follows. When 4 of the12 patients per treatment group have completed 5 days of daily dosing onPart B and have not experienced a serious drug-related adverse event,enrollment into the next higher dose level will occur, afterconsultation between the sponsor and investigators.

The first four patients enrolled will be assigned to the 50 mg dosegroup. If no serious drug-related adverse events are experienced, thenext 8 subjects will be randomized to either the 50 mg or 100 mg doselevel in a 1:1 fashion. If no serious drug-related adverse events occurin patients receiving 100 mg, then the next 24 patients will berandomized to either the 50, 100, or 200 mg dose group in a 1:2:3fashion.

If 4 of the 12 patients in a dose group experiences a seriousdrug-related event (Grade III or IV), 2 additional patients will beenrolled at the same dose level. Additionally, patient enrollment on tothe next dose level, if enrolling, will be temporarily on hold untilsafety is assessed. If one of the 2 additional patients experiences aserious drug-related event, dosing in this dose level will discontinue.Upon consultation with the sponsor and investigators, additionalpatients may be enrolled at a dose between the dose-limiting group andthe next lower dose group to determine the maximum tolerated dose (MTD).Enrollment of additional patients at a specific dose level will bedetermined in a protocol amendment.

The results indicated that a single 50 mg or 100 mg dose of BrEAincreased the numbers of activated CD8⁺ and CD4⁺ T cells (e.g., CD8⁺,CD69⁺, CD25⁻ cells) that were circulating in the patient's blood. Also,the circulating numbers of dendritic precursor cells, NK cells, LAKcells and cells that mediate ADCC (antibody-dependent cell-mediatedcytotoxicity mediated by the CD8⁺, CD16⁻ immune cell subset) functionswere increased. Further increases were usually observed on dosing for 5consecutive days.

Some of the results are summarized below. Course 1, 2 and 3 refer toeach 5 consecutive day treatment regimen of one daily injection withBrEA (50 or 100 mg BrEA per injection). The formulation contained 100mg/mL BrEA, PEG300-30% v/v, propylene glycol 30% v/v, benzyl benzoate30% v/v and benzyl alcohol 2% v/v. The data shown below was obtainedfrom patient blood samples at baseline (on the day dosing was initiated)and at various times after the patients received at least one dose ofBrEA. The results showed significant increases in immune cellpopulations and cytokine expression profiles associated with Th1responses. The patients in this protocol initially had CD4 counts of atleast 200 per mm³ and a serum HIV RNA load of 5,000 to 1×10⁶ RNAcopies/mL. After dosing with one course of BrEA (5 consecutive dailyi.m. injections), all patients showed increases in levels of immunecells including activated CD8 T cells (e.g., CD8⁺, CD69⁺, CD25⁻), LAKcells (e.g., CD8⁺, CD16⁺, CD38⁺), NK cells (e.g., CD8⁻, CD16⁺), ADCCcells (e.g., CD8⁻, CD16⁺) and dendritic cells (Lin⁻, HLA-DR⁺, CD11c⁺ orLin⁻, HLA-DR⁺, CD123⁺). Average CD4 IL-10 production dropped from amedian of 66% to 4% of the cells, while CD4 IFNγ went from a median of8% to 63%, leading to a Th2 to a Th1 shift in cytokine production.

In the tables below, baseline data is indicated by “BL” or by “pre”.

Increased immunophenotypes after BrEA therapy Phenotype Baseline^(a)Course 1 Course 2 Course 3 CD8+CD69+CD25− 18 54 56 75 n = (13) (13)  (9) (4) ^(b)p =    <0.001    <0.001    0.04 CD8+CD16+CD38+  8 27 28 25 n =(10) (10) ( 4)  (4) p =    <0.001    0.047    0.02 CD8−CD16+ 53 253 288  249  n = (12) (12)  (4)  (2) p =    <0.001    0.02    0.04Lin−HLA−DR+   3.2   17.7  11.4^(c)  14.7^(c) CD11c+/CD123+ n = (10) (10) (5)  (4) p =    <0.001    0.02    0.04 ^(a)Median values of cells/μL^(b)paired value t test ^(c)Test not available at baseline for patientsreceiving second and third courses, baseline value from initiation of2^(nd) course = 6.4 ^(d)% of CD4 ^(e)Baseline values from day 8(preceding the first five-day treatment)

Median activated T cells (CD8⁺ CD69⁺ CD25⁻ cells), LAK cells (CD8⁺CD16⁺CD38⁺), NK (ADCC responders) cells (CD8⁻ CD16⁺), dendritic cells(Lin⁻HLA-DR⁺CD123⁺/CD11⁺), and cells that mediate Th1 immune responses(IFNγ⁺ white blood cells) compared to baseline cell counts for 316α-bromoepiandrosterone treatments or treatment courses in theHIV-infected patients gave the following results. The results shownbelow for the treated patients were obtained about 1 week after the lastdose of 16α-bromoepiandrosterone was administered.

Activated T cells (CD8⁺ CD69⁺ CD25⁻) cells/μL Treatment 1 baseline 19treated 54 (n = 13, p < 0.001) Treatment 2 baseline 19 treated 56 (n =9, p < 0.001) Treatment 3 baseline 18 treated 74 (n = 4, p = 0.04)

LAK cells (CD8⁺ CD16⁺ CD38⁺) cells/μL Treatment 1 baseline  8 treated 27(n = 10, p < 0.001) Treatment 2 baseline 12 treated 28 (n = 4, p = 0.04)Treatment 3 baseline 12 treated 25 (n = 4, p = 0.02)

NK (ADCC responders) cells (CD8⁻ CD16⁺) cells/μL Treatment 1 baseline 54treated 253 (n = 12, p < 0.001) Treatment 2 baseline 58 treated 285 (n =4, p = 0.02) Treatment 3 baseline 56 treated 250 (n = 2, p = 0.04)

Dendritic cells (Lin⁻ HLA-DR⁺ CD123⁺/CD11⁺) cells/μL Treatment 1baseline 3.5 treated 18 (n = 10, p = 0.001) Treatment 2 baseline 6.5treated 11.5 (n = 5, p = 0.02) Treatment 3 baseline 6   treated 55 (n =4, p = 0.04)

These results show that the compound enhances the proportion ofcirculating cells that mediate cytotoxic immune responses and Th1 immuneresponses.

Treatment of HIV infected patients normalized their IL-10 producing CD4⁺T cells. In the same patients, 16α-bromoepiandrosterone was shown toenhance the proportion of CD4⁺ T cells that express detectable IFNγ.CD4⁺ IL-10⁻ IFNγ⁺ T cells mediate Th1 responses. These results show thatthe compound reduced the Th2 component of the immune system and enhancedthe Th1 component.

The results given above are a preliminary analysis based on dataobtained from 13 patients. Additional data was obtained for the patientsas follows.

CD4 counts were assessed using a FACScount (BDIS), and viral loads weremeasured using the bDNA assay (Bayer). Quality assurance procedures wereadhered to and internal and external controls were used to validate eachbatch of samples. On some occasions plasma viral loads were performed onbatched samples between cycles of dosing.

Whole blood was labeled with a cocktail of four monoclonal antibodies(Becton Dickinson Immunocytometry Systems, BDIS, San Jose, Calif.) percell subset using allophycocyanin, phycoerytherin, PerCP and FITCconjugates to measure surface phenotypes using a FACSCalibur (BDIS). Inaddition, four-color immunofluorescent analysis of blood cells wasperformed on the same schedule. The four color panels consisted of APC,PerCP, FITC, and PE reagents respectively in the following combinations:memory/naïve T cells (CD3/CD8/CD45RA/CD62L, CD3/CD4/CD45RA/CD62L), Tcell activation (CD3/CD8/CD69/CD25, CD3/CD4/CD69/CD25,CD3/CD8/HLA-DR/CD38, CD3/CD4/HLA-DR, CD38), B cell, LAK and NK(CD19/CD8/CD16/CD38) and dendritic cells (CD11c/HLA-DR/lineage markersCD3, CD16, CD14, CD19, CD56/CD123). Listmode data (25,000 to 50,000events) were analyzed using FCS Express (De Novo Software, Thornhill,Ontario, Canada). CD3⁺ cell subsets were identified by serial gating of(1) nucleated cells, (2) lymphocyte/lymphoblastoid cells, and (3) CD3+cells, followed by the gating of the subset of interest. For example,the absolute frequency per μL of any CD4+ cell subset (S) was estimatedby the equation: S=(proportion of CD4+ cells)×(CD4+ cell frequency perμL).

The CD4⁺ cell frequency was determined using the FACSCount test. Theabsolute frequency of a CD8⁺ cell subset was calculated in a similarfashion. The absolute white blood cell (WBC) count was determined usingan automated cell counter (Advia 120, Bayer, Tarrytown, N.Y.). Thenucleated cell region was defined using a forward versus orthogonalscatter plot. The absolute frequency per μL of CD3⁻ Natural Killer orDendritic Cell subsets (F) was estimated by the equation: F=(proportionof nucleated cells)×(absolute WBC count per μL).

Statistical analysis of changes in surface phenotypes, hematological,and viral parameters were performed by calculating the area under thecurve (AUC) of the percentage difference from baselines (phenotype),percentage difference from the mean of screen and baseline values(hematology), or log₁₀ change from the mean of screen and baselinevalues (plasma viral RNA) for individual patients. AUC were calculatedusing the actual number of days between analysis visits for eachpatient. Because patients were on study for varying periods of time,this value was normalized to the time-averaged AUC by dividing by thenumber of days of observation. The Student's t-test was then used toanalyze the difference from zero for the AUCs from all patients withdata available for each parameter. A significant increase in the AUC forthe entire 5-month period was interpreted as a nonrandom change in thegiven parameter.

Changes occurred soon after the intermittent dosing for a number of themeasurements, and patients showed variability in the kinetics ofimmunologic, hematologic and virologic responses. For these reasons, thetime-averaged AUC values for the 5-month period underestimate themagnitude of the individual responses. Maximal individual patientresponses were calculated as the mean of the maximal percentage changesfrom baselines for individual patients at any analysis visit afterdosing.

To investigate the kinetics of the responses for the entire populationrelated to the time after dosing, the maximum analysis visit responseswere calculated as the average of the maxima of the mean percentagechanges from baselines for each analysis visit. Due to the altered BrEAadministration interval for cohort 3, the analysis visits were alignedrelative to the last dosing visit.

BrEA treatment of the HIV infected patients caused changes in T cell anddendritic cell phenotypes that persisted for weeks after intermittentdosing was discontinued. Increases were observed after dosing in boththe total activated and early activated stage CD8⁺ T cells as well as incirculating total dendritic cells consisting of both the CD11c⁺ and theCD123⁺ phenotypes. The significant increases observed in the AUCanalyses over the entire 5-month intermittent dosing period indicatethat these changes are not random fluctuations. Although activation ofCD8⁺ cells is linked with disease progression, intact dendritic cellfunction and activation of CD8⁺ T cells is also requisite forstimulation of CTL, both for specific anti-HIV-1 responses, as well asfor anti-opportunistic infection responses. In HIV infection there is aprogressive decrease in circulating CD4⁺ T cells, an increase incirculating CD8⁺ T cells, and a decrease in the CD4/CD8 ratio. In thisstudy, the decreases in both circulating CD4⁺ and CD8⁺ T cells with nochange in the CD4:CD8 ratio may indicate that activated T cells aretrafficking to sites of infection in the lymph nodes and mucosal tissue.

In this study, there was no overall decrease in viral load after three5-day courses of intermittent dosing over a period of 153 days. It ispossible that a CTL-driven elimination of HIV-1 infected cells withlong-term effects on viral levels may result from continued dosing orfrom adjustments in the route of administration or the dose-schedule.

Treatment with BrEA resulted in significant changes in hematologyparameters in the patients including increased circulating platelets (anaverage 20% increase over baseline on study day 126), monocytes (anaverage 49% increase over baseline on study day 50) and neutrophils (anaverage 51% increase over baseline on study day 81). These changes maydue to stimulation of hematopoiesis or redistribution of thehematopoietic elements from the tissues to the circulation. The durationof the increases in neutrophils and platelets that have in vivohalf-lifes of approximately 10 and 107 hours respectively suggest thatat least part of the observed responses is due to enhanced hemopoiesisin these patients.

Some of the data that was obtained is summarized below.

Timing of BrEA Dosing and Analyses Dosing Visit Analysis Visit MedianDays After Last Dose Days^(A) Days^(B) to Analysis Visit (Range)^(C) 1 77 (7-11) 15-19 22 3 (2-10) 36 17 (13-24) 50 31 (26-36) 60-64 67 3 (0-5) 81 17 (13-19) 95 31 (25-33) 118-122 126 3 (0-5)  139 17 (12-23) 153 31(26-33) ^(A)Aligned study days on which patients received injections ofBrEA. ^(B)Aligned study days on which analysis samples were taken.^(C)The median number and range of days after patients received the lastdose of BrEA prior to the analysis visit.

For the following table, the symbols in the table have the meaningsgiven here. ^(A)Number of patients with data for phenotype, dendriticcell analysis was not initiated at start of study. ^(B)Mean increase inthe time-averaged individual AUC differences from baseline values forall patients. ^(C)p value for Student's t-Test for individual AUCpercentage increases from baseline. ^(D)Maximal individual patientresponses were calculated as the mean of the maximal percentage changesfrom baselines for individual patients at any analysis visit afterdosing. The range of responses is indicated in parentheses. ^(E)Maximumanalysis visit responses were calculated as the average of the maxima ofthe mean percentage changes from baselines for each analysis visit. Theday of maximal response is indicated in parentheses. ^(F)CD3⁺ CD8⁺ andCD69⁺ or CD25⁺. ^(G)CD3⁺ CD8⁺ CD69⁺ D25⁻. ^(H)CD8⁻ CD16⁺. ^(I)CD8⁺CD16⁺. ^(J)Lineage⁻ HLA-DR⁺ CD11c⁺. ^(K)Lineage⁻ HLA-DR⁺ CD123⁺.

Immune Phenotype Changes after BrEA Intramuscular Administration MaximumMaximum Analysis Visit Time- Individual Responses averaged t-TestPatient Mean AUC Increase p- Responses (analysis visit Phenotype n^(A)Mean ± SEM^(B) value^(C) Mean (range)^(D) day)^(E) Activated CD8⁺ T 3615 ± 5%  0.005  70% (−10 to 340%) 25% (22) cells^(F) Early Activation-36 67 ± 33% 0.049 210% (0 to 3.090%) 162% (153) Stage CD8⁺ T cells^(G)NK^(H) 36 29 ± 22% 0.20 140% (−40 to 1.220%) 41% (36) LAK^(I) 36 13 ±9%  0.19  90% (−90 to 630%) 34% (67) Total Dendritic Cells 31 38 ± 14%0.010 100% (−20 to 600%)  38% (139) DC1 (CD11c⁺) 31 50 ± 18% 0.010 130%(−20 to 870%) 58% (22) Dendritic Cells^(J) DC2 (CD123⁺) 31 32 ± 13%0.021 100% (−30 to 480%) 44% (80) Dendritic Cells^(K) DC1 to DC2 Ratio31 44 ± 13% 0.003

Increase In Circulating Hematologic Elements After Intramuscular BrEAAdministration Time- averaged Maximum Maximum AUC Individual AnalysisVisit Increase Patient Responses Mean Hematologic Mean ± t-TestResponses (analysis visit Elements n^(A) SEM^(B) p-value^(C) Mean(range)^(D) day)^(E) WBC 37 4.4 ± 3%   0.132  36% (−1 to 159%) 23% (81)Neutrophils 37 11 ± 4%  0.009  67% (−25 to 297%) 51% (81) Eosinophils 3320 ± 11% 0.086 110% (−37 to 656%) 129% (22)  Basophils 37 42 ± 12% 0.001182% (−20 to 1.100%) 113% (126) Monocytes 37 10 ± 5%  0.029  60% (−14 to189%) 49% (50) Platelets 37 7 ± 2% <0.001  31% (0 to 122%)  20% (126)^(A)Number of patients with data for hematologic element. ^(B)Meanincrease in the time-averaged individual AUC differences from baselinevalues for all patients. ^(C)p value for Student's t-Test for individualAUC percentage increases from baseline. ^(D)Maximal individual patientresponses were calculated as the mean of the maximal percentage changesfrom baselines for individual patients at any analysis visit afterdosing. The range of responses is indicated in parentheses. ^(E)Maximumanalysis visit responses were calculated as the average of the maxima ofthe mean percentage changes from baselines for each analysis visit. Theday of maximal response is indicated in parentheses.

In a separate clinical trial using 100 mg of BrEA delivered to patientsonce per day for 5 consecutive days by intramuscular injection, severalpatients were evaluated for changes in the ratio of CD4⁺ memory T cell 1cells (intracellular IFNγ⁺ CD45RA⁻ CD62L⁻ CD11a^(bright)) or MT1 cellsto memory T cell 2 cells (intracellular IL-4⁺ CD45RA⁻ CD62L⁺CD11a^(dim)) or MT2 cells. MT1 cells mediate or facilitate Th1 immuneresponses and MT2 cells mediate or facilitate Th2 immune responses. Anincrease in the MT1:MT2 ratio indicates an enhanced Th1 immune responseor immune status. See, e.g., D. K. Mitra et al., InternationalImmunology 1999 11:1801-1810. The tested patients (7/7) showed atransient increase in the MT1:MT2 ratio after a 5 day course of dosingwith BrEA. The maximum observed increase was about 700% in one patientat 10 days after the last dose of BrEA was administered. The increaseusually persisted for more than 10 days after the last dose of BrEA wasadministered. These results showed that BrEA was capable of enhancingthe numbers of circulating immune cell subsets that mediate Th1 typeresponses.

Example 21

Treatment of symptoms of HIV infection. Two HIV infected patients withchronic diarrhea were dosed with BrEA as follows. A BrEA formulation (40mg/mL BrEA in 25% v/v PEG 300, 12.5% v/v ethanol, 5% v/v benzylbenzoate, ˜57.5% v/v propylene glycol) was delivered subcutaneously. Thepatients received 60 mg of BrEA in 1.5 mL daily for 10 days. During theperiod of dosing, the diarrhea ceased. After the 10-day dosing periodended, diarrhea resumed. In other patients receiving oral BrEA, diarrheaalso went into remission.

Example 22

Subcutaneous formulation. A BrEA formulation was prepared essentially asdescribed herein. The formulation contained 50 mg/mL BrEA, 40% v/v PEG200, 2% v/v benzyl alcohol, 2% v/v benzyl benzoate and ˜66% v/vpropylene glycol (qs). The formulation is particularly suitable forsubcutaneous administration of the compound.

Example 23

Preparation of BrEA hemihydrate-procedure 1. Crude BrEA was prepared bybromination of epiandrosterone, followed by crystallization frommethanol. The hemihydrate was prepared by dissolving 25 g of crude BrEAin 75 mL of refluxing ethanol with moderate agitation. To the BrEAsolution 12.5 mL of water was slowly added while maintaining thesolution at reflux with agitation. Agitation of the solution wasmaintained and the solution was then allowed to cool to about 20-25° C.and kept at about 20-25° C. for about 15 minutes to obtain a suspensionof BrEA hemihydrate crystals. The crystals were recovered by filtration,washed with a solution of 25 mL of water:ethanol (5:1 v/v) at about20-25° C. and then vacuum dried for about 13 hours at 50-60° C. untilthe product weight was constant. The crystals were primarily rod andneedle shaped, with smaller amounts of other shapes such as tablets.

The procedure gave 22.5 g of BrEA hemihydrate (yield 90%) with a watercontent of 2.6% w/w by KF analysis, a purity of 100% by HPLC areaanalysis, an FTIR spectrum with carbonyl peaks at 1741 cm⁻¹ and 1752cm⁻¹. The FTIR scan of anhydrous BrEA shows a single carbonyl peak at1749 cm⁻¹. The DSC scan showed three endotherms. One had a broad shallowpeak with an onset at about 109-110° C. and ending at about 150° C. Thisbroad DSC peak is consistent with the loss of water from the hemihydratecrystals as the temperature of the sample increased. The secondendotherm at about 83-100° C. is consistent with the loss of the smallamount of residual ethanol from the sample. A DSC scan of anhydrous BrEAdoes not have the broad endotherm that is observed with the hemihydrate.Also consistent with the loss of water from the hemihydrate over the100-150° C. range is a sharp third endotherm peak in the hemihydrate DSCscan at about 163-164° C., which is the melting point of anhydrous BrEA.The FTIR was obtained using USP method <197>, where the BrEA hemihydratesample was prepared in KBr. The DSC thermogram was obtained by scanningfrom 25° C. to 250° C. with a heating rate of 10° C./minute.

Example 24

Preparation of BrEA hemihydrate—procedure 2. The hemihydrate wasprepared by dissolving 10 g of crude BrEA in 40 mL of refluxing acetonewith moderate agitation. To the BrEA solution 4.0 mL of water was slowlyadded while maintaining the solution at reflux with agitation. Agitationof the solution was maintained and the solution was then allowed to coolto about 20-25° C. and kept at about 20-25° C. for about 15 minutes toobtain a suspension of BrEA hemihydrate crystals. The crystals wererecovered by filtration, washed with a solution of 6.0 mL ofwater:acetone (10:1 v/v) at about 20-25° C. and then vacuum driedovernight (about 13-15 hours) at 50-60° C. until the product weight wasconstant. The procedure gave 7.0 g of BrEA hemihydrate (yield 70%) witha water content of 2.6% w/w by KF analysis and an FTIR spectrum withcarbonyl peaks at 1741 cm⁻¹ and 1752 cm⁻¹.

Example 25

Analysis of BrEA hemihydrate particle size. BrEA hemihydrate crystalswere prepared essentially as described herein and sized using a particlesizing apparatus (Malvern Instruments). The analysis model used was fora polydisperse sample and a volume distribution type. The analysisshowed a range of crystal diameter sizes from about 0.5 μm to about 880μm. About 90% of the crystals had a diameter of about 20 μm to about 220μm and the majority of the crystals had a diameter of about 30-200 μm.The mean crystal diameter was about 93 μm. The specific surface area ofthe crystals was about 0.25 m²/g.

Example 26

Formulations containing BrEA hemihydrate were prepared. Similarformulations using other formula 1 compounds are prepared using the sameor similar excipients, e.g., a different preservative can be used in thesuspension formulation instead of methylparaben.

A formulation containing BrEA hemihydrate in aqueous suspension wasprepared. The BrEA hemihydrate had an average particle size of less than20 μm and it was mixed with polysorbate 80 before addition to the liquidcomponents. The final aqueous composition contained 100 mg/mL BrEA, 2%w/v polysorbate 80, 0.1% w/v carboxymethylcellulose sodium, 0.82% w/vsodium chloride, 0.023% w/v dibasic sodium phosphate, 0.101% w/vmonobasic sodium phosphate, 0.5% v/v ethanol and 0.1% w/v methylparaben,pH 6.5+/−0.4. The formulation was prepared using sterile technique. Thisformulation is suitable for subcutaneous, intramuscular orintraperitoneal delivery of BrEA, which can be delivered in a bolus ordepot in the skin, muscle, peritoneal cavity or other suitable site in asubject. The formulation is not generally used for intravenous delivery,particularly in humans, due to the presence of the drug particles.

A caplet (capsule shaped tablet) containing BrEA hemihydrate wasprepared using compressible sucrose. The caplets each contained 25 mgBrEA hemihydrate, 6.25 mg povidone (1-ethenyl-2-pyrrolidinone polymer),0.62 mg magnesium stearate, 45 mg mannitol and 48.12 mg of compressiblesucrose. Sterile BrEA and excipients were used to prepare the caplets.The formulation is suitable for buccal or sublingual, delivery of BrEA(or another formula 1 compound) to a subject.

Example 27

Inhibition of inflammation. The capacity of formula 1 compounds to limitor inhibit inflammation or symptoms of inflammation was shown usinganimal models for inflammatory bowel disease.

Groups of 3 male Wistar rats (180±20 grams) fasted for 24 hours before2,4-dinitrobenzene sulfonic acid (DNBS) or saline challenge were used.Distal colitis was induced by intra-colonic instillation of 0.5 mL of anethanolic solution of DNBS (30 mg in 0.5 mL of a 30% ethanol in salinesolution) after which 2 mL of air was injected through the cannula toensure that the solution remained in the colon. The volume used was 0.1mL per injection of 2 and 20 mg/mL of 7-oxodehydroepiandrosterone in aliquid formulation, was administered by subcutaneous injection once aday for 6 days. The formulation contained 100 mg/mL of7-oxodehydroepiandrosterone in a non-aqueous suspension that contained2% benzyl alcohol w/v, 0.1% Brij 96 w/v and equal volumes of PEG 300 andpropylene glycol. Concentrations of 2 mg/mL and 20 mg/mL were obtainedby diluting the 20 mg/mL formulation with vehicle that lacked the activesubstance.

The first dose was given 30 minutes after DNBS challenge. Sulfasalazine(30 mg/mL in 2% Tween 80 in distilled water) was administered orally(PO) once a day (10 mL/kg/day) for 7 days, the first two doses beginning24 hours and 2 hours before DNBS challenge. The presence of diarrhea wasrecorded daily by examining the anal area. Animals were fasted for 24hours prior to being sacrificed. Animals were sacrificed on day 7 or day8 and their colons were removed and weighed. Before removal of thecolon, signs of adhesion between the colon and other organs wererecorded. Also, the presence of ulcerations was noted after weighing ofeach colon. The “net” change of colon-to-body weight (BW) ratio wasnormalized relative to saline-challenged baseline group. A 30% decreasein “net” colon-to-body weight ratio was considered significant.

A total of five studies were conducted. Data from several studies arecombined. The 7-oxodehydroepiandrosterone at 2 and 20 mg/mL, decreasedthe net colon-to-body weight ratio by 19 and by 14% relative tovehicle-treated group, respectively. Adhesions were absent in all threetested animals. Colonic ulceration was noted in ⅓ animals. All animalshad diarrhea. Similarly, all animals treated with Sulfazalazine haddiarrhea. Sulfasalazine exhibited significant protection frominflammation (−33% change in net colon-to-body weight (BW) ratiorelative to EE-1 treated group) and no animals exhibited adhesions orcolonic ulceration. The formula 1 compound3β,7β,17β-trihydroxyandrost-5-ene (AET) was administered by subcutaneousinjections of 0.2 and 2.0 mg/day for 5 or 6 days, with the first doseinjected 30 minutes post-DNBS challenge. The AET formulation was thesame as the 7-oxodehydroepiandrosterone formulation, except that AETreplaced 7-oxodehydroepiandrosterone. AET decreased the netcolon-to-body weight ratio by 15% and 21% relative to controls (see thetable below). Animals treated with AET (2 mg) had no diarrhea. Adhesionwas absent and fewer animals had colonic ulcerations (33% relative tountreated animals, N=9, pooled data from two studies). The sameformulations containing BrEA (100 mg/mL) or dehydroepiandrosterone (20mg/mL) decreased the net colon-to-body weight ratio by 21 and 3%,respectively relative to controls. In four subsequent studies, AETvariably reduced the net colon-to-body weight ratio, ulcers anddiarrhea.

Severe acute inflammation, measured 7 days after DNBS challenge, wasobserved in 3 of 10 immunosteroid vehicle control animals, while AET,dehydroepiandrosterone, sulfasalazine, and sulfasalazine vehicle(polysorbate 80) resulted in severe inflammation in 4, 10, 3, and 8animals respectively. Moderate acute inflammation, measured 7 days afterDNBS challenge, was observed in 5 of 10 immunosteroid vehicle controlanimals, while AET, dehydroepiandrosterone, sulfasalazine, andsulfasalazine vehicle (polysorbate 80) resulted in moderate inflammationin 6, 0, 6, and 2 animals respectively. Severe inflammation at day 7 wasobserved in 3, 4, 10 and 3 animals that were treated with vehicle, AET,dehydroepiandrosterone and sulfasalazine respectively. Severe chronicinflammation, measured 14 days after DNBS challenge, was observed in 8of 10 immunosteroid vehicle control animals, while AET,dehydroepiandrosterone, sulfasalazine, and sulfasalazine vehicle(polysorbate 80) resulted in severe inflammation in 3, 3, 2, and 6animals respectively.

At day 7 (acute inflammation) after DNBS challenge, 8 of 10 animalstreated with vehicle control had severe ulceration, while 2 of 10animals treated with AET had severe ulceration. Such ulceration wasobserved in 3 of 10 sulfasalazine control and in 10 of 10 animalstreated with dehydroepiandrosterone. Delaying the initiation of AETtreatment until 2 days after DNBS challenge resulted in increasedchronic ulceration, with 5 of 10 animals showing ulcers 14 days afterchallenge, compared to only 1 animal with ulcers when treatment beganthe same day as challenge. This indicated that for acute inflammation inthis model, AET is most effective early in the initiation of a flare ofinflammatory cellular responses. At day 14 after DNBS challenge (chronicinflammation) 5 of 10 vehicle controls showed severe ulceration, 1 of 10AET treated animals had severe ulceration, 0 of 10 animals treated withdehydroepiandrosterone had severe ulceration and 4 of 10 sulfasalazinetreated animals (positive control) has severe ulceration. The resultsshowed that the formula 1 compounds reduced inflammation or its symptomscompared to untreated control animals.

In another protocol formula 1 compounds were used in a similar animalmodel. Groups of 10 male Sprague Dawley rats (250-300 grams) were fastedfor 24 hours before 2,4,6-trinitrobenzene sulfonic acid (TNBS) or salinechallenge. This model presents a severe challenge to the animal's immunesystem. On Day 0, the rats were lightly anesthetized and a catheterinserted rectally into the colon such that the tip was 8 cm proximal tothe anus. Distal colitis was induced by intra-colonic instillation of0.5 mL of an ethanolic solution of TNBS (60 mg/mL ethanol 50% in water).The test substance, 7-oxodehydroepiandrosterone 20 mg/mL in vehicle orvehicle control was administered by subcutaneous injection (0.1mL/injection) once a day for 6 days, the first dose being given 30minutes after TNBS challenge. Sulfasalazine (30 mg/mL in 2% Tween 80 indistilled water) or vehicle control was administered orally (PO) once aday (10 mL/kg/day) for 7 days, the first two doses beginning 24 hoursand 2 hours before DNBS challenge. Signs of diarrhea were recorded dailyby examining the anal area. Animals were fasted for 24 hours prior tosacrifice on day 14 and the colon was removed and weighed. After grossobservations of colonic tissues, 3 samples were taken from regionsapproximately 1, 3, and 8 cm proximal to the anus. If gross ulcers orinflammation were present, at least one sample was taken from theaffected region. Before removal of the colon, signs of adhesion betweenthe colon and other organs were recorded. Also, the presence of coloniculcerations and intestinal adhesions was noted after weighing of eachcolon. Body weight, colon-to-body-weight ratio, and damage scores wereassessed. Visible damage was scored on a 1-5 scale as follows: 0=Nodamage; 1=Localized hyperemia but no ulcers; 2=linear ulcers with nosignificant inflammation; 3=Linear ulcers with inflammation at one site;4=Two or more sites of ulceration and/or inflammation; 5=Two or moremajor sites of ulceration and inflammation or one major site ofulceration and inflammation extending more than 1 cm along the length ofthe colon. Inflammation is defined as regions of hyperemia and bowelwall thickening.

Ulcerations were present more frequently at multiple colonic levelsfollowing 7-oxodehydroepiandrosterone treatment compared to controls.Moderate to severe ulcerative lesions associated with moderate to severefibrosis of the mucosa and/or submucosa occurred in 8/9 (89%) of7-oxodehydroepiandrosterone treated animals compared to 5/9 (56%) ofcontrols. The severity and frequency of muscular, peritoneal and/ormesenteric inflammation appeared slightly more than in controls. Thesehistopathological data suggests that 7-oxodehydroepiandrosteroneexacerbated the inflammation. Moderate to severe ulcerative lesionsoccurred in 6/10 (60%) and 5/10 (50%) of sulfazalazine and vehicletreated animals, respectively. The severity and frequency of muscular,peritoneal and/or mesenteric inflammation appeared to be similar to thatoccurring in vehicle groups. The frequency of moderate to severe lesionsand their severity based on their occurrence at multiple colonic levelsappeared to be similar between the negative control (vehicle), referencecontrol (sulfazalazine), BrEA and dehydroepiandrosterone, indicatingthat for these compounds the degree of inflammation was similar for thecontrol compound and the tested compounds.

Example 28

Modulation of delayed type hypersensitivity.3β,7β,17β-trihydroxyandrost-5-ene (AET) was examined for its capacity tomodulate delayed type hypersensitivity (DTH). Groups of five femaleBALB/cByJ mice (20-25 grams) were anesthetized and 100 μL of a 3%solution of oxazolone was applied on day 0 to the shaved abdomen anddried. Seven days later, on day 7, the mice were challenged by applying5 μL of oxazolone topically to each side of the right ear. The compoundAET (40 mg/mL) in vehicle was administered by subcutaneous injection (2mg/day, 50 μL/injection) one time on day 6, 24 hours before theoxazolone challenge. The vehicle as a non-aqueous suspension of AET in2% benzyl alcohol w/v, 0.1% Brij 96 w/v and equal volumes of PEG 300 andpropylene glycol.

Dexamethasone in saline (0.2 mg/mL) was administered daily for 9 days(day −1 to 7), first dose 24 hours before sensitization, last dose atchallenge by subcutaneous injection (0.01 mg/dose, 50 μL/injection). OnDay 8, 24 hours following the oxazolone challenge, both the right andleft ear thicknesses were measured using a micrometer caliper and thedifferences determined. The differential ear thickness is measured as anindicator of the DTH response to topical oxazolone challenge. The DTHresponse was expressed as the difference in the thickness (mm) betweenthe right and the left ears for each animal.

The differential ear thickness in animals receiving vehicle alone was0.225 mm and treatment with dexamethasone (high dose) orcyclophosphamide reduced the DTH response (0.144 mm and 0.092 mm,respectively). AET administered subcutaneously had only a slight effecton the DTH response to oxazolone. When administered 24 hours beforechallenge or at challenge (2 mg/day), the effect was a 22-24% reductionin the response. When administered daily for 8 days (2 mg/day), firstdose 24 hours before sensitization and last dose 24 hours prior tochallenge, the effect was an enhancement of the response (23%). Thisresult shows that if the compound was delivered to the animals duringsensitization, the DTH response increased. This is consistent with anenhanced Th1 immune response. If the compound was delivered to theanimals after sensitization, the DTH response was decreased. This isconsistent with a decreased inflammatory response.

Example 29

Reversal of immunosenescence. Healthy aged (20-month) orimmunologically-mature (3-month) BALB/c mice were vaccinated withhepatitis B surface antigen (HBsAg) (2 μg; Recombivax-HB; Merck) andAlum (2.75 μg). The aged mice were vaccinated with the antigen and alsoreceived a single subcutaneous injection of either 0.3 mg or 3.0 mg of3β,7β,17β-trihydroxyandrost-5-ene (AET), BrEA or7-oxodehydroepiandrosterone, dehydroepiandrosterone or the vehicle(placebo control). The AET, BrEA hemihydrate or dehydroepiandrosteronewas present in a formulation that contained 60 mg/mL of the compoundsuspended in 0.1% w/v carboxymethyl cellulose sodium in 0.9% saline.7-Oxodehydroepiandrosterone was in a formulation that contained 60 mg/mLof the compound suspended in 0.1% w/v gum arabic in 0.9% saline.

A concentration of 6 mg/mL was obtained by diluting the 60 mg/mLformulation with vehicle that lacked the active substance.

Blood samples were collected 14, 21 and 34 days after treatment and thesera were analyzed by ELISA to determine the concentration ofHBsAg-specific IgG (total IgG). In addition, samples obtained on day 21were analyzed to determine the concentration of HBsAg-specific IgG1 andIgG2a subclasses. The results summarized below were average valuesobtained with blood samples collected 21 days after vaccination ofgroups of 8 mice. Subcutaneous injection was performed after shaving thehair from the thighs of each mouse. The injected volume was 50 μL forcompound (3.0 mg or 0.3 mg) or placebo, and for vaccine preparation. Thevehicle control consisted of carboxymethylcellulose (0.5%) in saline(0.9%). Antibody titers were determined by ELISA.

Treatment of aged, vaccinated animals with the formula 1 compounds,resulted in higher anti-HBsAg IgG titers than aged animals receiving thevaccination only. Higher antibody titers were achieved in aged mice thatreceived the compounds at the same time as vaccination, in the majorityof cases. For all tested compounds, except for BrEA at the low dose (0.3mg), the IgG titer increase was significant compared to aged controls.This result shows that the formula 1 compounds result in an enhancedimmune response to antigen challenge in the immune senescent animals.

The serum samples were also analyzed for the titers of HBsAg-specific,IgG1 or IgG2a immunoglobulin subclasses. A bias to IgG1 is seen in agedmice and this is considered symptomatic of immune senescence or asuboptimal immune response associated with immune senescence. TheIgG1/IgG2a ratio is an indicator of immune status. Th2 cellspredominantly assist in the generation of humoral immunity, while Th1cells enhance, e.g., cellular immunity. Humoral immunity (Th2) becomespredominant with age, while the decreasing cellular (Th1) immunity leadsto increased susceptibility to, e.g., infectious diseases.

The ratio of IgG1 to IgG2a of about 8:1 was measured in the young miceand 27:1 in aged mice. Generation of antigen-specific IgG1 generationinvolves T-helper type 2 (Th2) cells, and for IgG2a, T-helper type 1(Th1) cells. Treatment of aged animals with the formula 1 compoundsshifted the IgG1/IgG2a ratios toward the ratio seen in young, vaccinatedanimals. The ratios observed from the animals treated with AET, BrEA,7-oxodehydroepiandrosterone or dehydroepiandrosterone ranged from about4 to about 13. The shift was statistically significant. The testedcompounds all enhanced the proportion of IgG2a, and thus the associatedTh1 response to the antigen.

Secondary antibody response. Later, 42 days after the initial exposureto HBsAg, serum samples were taken from the mice described in theprevious section and these were tested for anti-HBsAg IgG. At thistime-point, vaccine-specific IgG titers were either low or undetectable.Three days later (45 days after first vaccination), the mice wereinjected again with HBsAg in Alum, but this time, none of the micereceived immunosteroid (secondary vaccination). Serum samples collected7 days and 14 days after the second exposure to HBsAg vaccine wereassayed for anti-HBsAg antibody. In the young mice, a marked increase inspecific antibody was seen in response to the second vaccination. Incontrast, in aged mice that had received no immunosteroid with the firstHBsAg injection, low levels of anti-HBsAg were detectable, and only in 4of the 8 mice in this group was antibody detectable. Increases inanti-HBsAg titers were seen following secondary vaccination in agedanimals that had been treated with immunosteroid in conjunction with thefirst HBsAg exposure. The anti-HBsAg antibody titer in serum increasedfollowing secondary vaccination in all 8 mice in the groups of aged micethat were treated at primary vaccination with the higher dose (3mg/mouse) of AET, BrEA or 7-oxodehydroepiandrosterone, or withdehydroepiandrosterone (0.3 mg/mouse). In those aged mice that hadreceived the lower dose (0.3 mg/mouse) of AET, BrEA or7-oxodehydroepiandrosterone at vaccination, an intermediate response wasseen, with 6 of the 8 mice producing detectable anti-HBsAg in responseto secondary vaccination. These results show that the tested compoundsresulted in an enhanced secondary antibody response in the aged animals.The compounds delivered by a transmucosal route (e.g., buccal orsublingual) can also provide an unexpectedly superior effect to thecompounds when they are delivered by other routes such as subcutaneousor oral.

Example 30

DNA vaccine adjuvant. Formula 1 compounds such as3β,7β,17β-Trihydroxyandrost-5-ene (AET) and BrEA are used to modulatethe immune response to an antigen(s) such as malaria antigens encoded byDNA expression vectors. Antigens such as a Plasmodium, e.g., P. yoelii,P. falciparum, P. vivax or P. berghei, circumsporozoite or merozoiteprotein are used to immunize a subject. AET or BrEA is administered onthe same day or a day or two before antigen challenge. Suitableantigens, expression vectors and their delivery to a subject have beendescribed. See, e.g., S. L. Hoffman et al., Vaccine 1994 12:1529-1533,R. Weiss et al., Infect. Immunity 2000 68:5914-5919, J. C. Rayner etal., Proc. Nat'l. Acad. Sci. U.S.A. 2000 97:9648-9653, S. Scheiblhoferet al., Eur. J. Immunol. 2001 31:692-298. The capacity of the compoundsto enhance immune responses to the antigens by, e.g., measuringcytotoxic T lymphocytes or antibody titer after delivery of the formula1 compound and immunization with an antigen(s). Typically the immuneresponse is measured at about 10 days to about 21 days after a primaryimmunization. Methods to measure immune responses are essentially asdescribed herein or in appropriate cited references. DNAs that encode anantigen(s) that is associated with, e.g., an infectious agent or a tumordescribed herein may be used in these assays. The capacity of theformula 1 compound to modulate the immune response(s) to antigenchallenge is optionally compared to the response generated by AET orBrEA.

Example 31

Modulation of monocyte or macrophage activation or survival. Thecapacity of the formula 1 compounds to activate monocytes and/orincrease monocyte or macrophage activity or survival is determined usingmethods known in the art. The formula 1 compounds are assayed using,e.g., the assays described below. For these assays, peripheral bloodmononuclear cells (PBMC) are purified from a subject, e.g., a humanleukopack (American Red Cross, Baltimore, Md.) by centrifugation througha histopaque gradient (Sigma). Monocytes are isolated from PBMC bycounterflow centrifugal elutriation. In each of the assays, the activityof a given formula 1 compound is optionally compared to the responseassociated with AET or BrEA.

Modulation of monocyte survival is determined essentially as follows.Human peripheral blood monocytes progressively lose viability whencultured in absence of serum or other stimuli. Their death results frominternally regulated process such as apoptosis. Addition to the cultureof activating factors, such as TNF-α improves cell survival. Propidiumiodide (PI) staining is used to measure apoptosis as follows. Monocytesare cultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of about 100 ng/mL of TNF-α(negative control), and in the presence of varying concentrations of theformula 1 compound. Cells are suspended at a concentration of 2×10⁶/mLin PBS containing PI at a final 5 concentration of about 5 μg/mL, andthen incubated at room temperature for 5 minutes before FACScananalysis. PI uptake has been demonstrated to correlate with DNAfragmentation in this experimental paradigm. The activity of formula 1compounds such as AET or BrEA can be used as a comparison standard forother formula 1 compounds.

The effect of formula 1 compounds on monocyte or macrophage cytokinerelease is determined essentially as follows. An important function ofmonocytes and macrophages is their regulatory activity on other cellularpopulations of the immune system through the cytokines release afterstimulation. An ELISA to measure cytokine release is performed using,e.g., human, monocytes, which are incubated at a density of about 5×10⁶cells/mL. A range of formula 1 compound concentrations is used, e.g., 1nm, 10 nm, 100 nm, 1 μm, 10 μm and 50 μm. To determine IL-12 production,the cells are primed overnight with IFN (100 U/mL) in presence of aformula 1 compound. LPS (10 ng/mL) is then added. Conditioned media arecollected after 24 h and kept frozen until use. Measurement ofTNF-alpha, IL-10, MCP-I and IL-8 is then performed using a commerciallyavailable ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)) andapplying the standard protocols provided with the kit.

Activation of monocytes or macrophages by formula 1 compound is measuredby assaying the oxidative burst after stimulation of the cells. Purifiedmonocytes are plated in 96-well plate at about 2×10⁵ cells/well. A rangeof formula 1 compound concentrations, e.g., 1 nm, 10 nm, 100 nm, 1 μm,10 μm and 50 μm, are added to the wells in a total volume of 0.2 mLculture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3days incubation, the plates are centrifuged and the medium is removedfrom the wells. To the macrophage monolayers, 0.2 mL per well of phenolred solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, togetherwith the stimulant (200 nM PMA). The plates are incubated at 37° C. for2 hours and the reaction is stopped by adding 20 μL 1N NaOH per well.The absorbance is read at 610 nm. To calculate the amount of H₂0₂produced by the macrophages, a standard curve of a H₂O₂ solution ofknown molarity is performed for each experiment.

Example 32

Modulation of central nervous system cell growth, differentiation oractivity. The formula 1 compounds are used to modulate astrocyte orneuron survival, neurite outgrowth, phenotypic differentiation ofcortical neuronal cells or for inducing the proliferation of glialfibrillary acidic protein immunopositive cells (astrocytes). A thymidineincorporation assay, for example, can be used to measure cellproliferation or survival. The biological effects of FGF-2 (basic FGF)on cortical or hippocampal neurons in vitro included increases in bothneuron survival and neurite outgrowth (Walicke et al., Proc. Natl. Acad.Sci. U.S.A. 1986 83:3012-3016. Using primary cortical neuronal culture,the effect of a formula 1 compound to induce neurite outgrowth can becompared to the response achieved with FGF-2 using, for example, athymidine incorporation assay.

The formula 1 compounds are used in a model for Parkinson diseaseessentially as follows. The loss of motor function in Parkinson'sdisease is attributed to a deficiency of striatal dopamine resultingfrom the degeneration of the nigrostriatal dopaminergic projectionneurons. An animal model for Parkinson's that has been extensivelycharacterized involves the systemic administration of 1-methyl-4 phenyl1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up byastrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenylpyridine (MPP) and released. Subsequently, MPP is actively accumulatedin dopaminergic neurons by the high-affinity reuptake transporter fordopamine. MPP is then concentrated in mitochondria by theelectrochemical gradient, which selectively inhibits nicotidamideadenine disphosphate; ubiquinone oxidoreductionase (complex 1), therebyinterfering with electron transport and eventually generating oxygenradicals. It has been demonstrated in tissue culture and in vivo thatFGF-2 (basic FGF) has trophic activity for nigral dopaminergic neurons.FGF-2 in gel foam implants in the striatum results in the near completeprotection of nigral dopaminergic neurons from the toxicity associatedwith MPTP exposure. The formula 1 compounds are administered with orwithout FGF-2 to measure their capacity to enhance survival ofdopaminergic neurons in vitro, or they are delivered in vivo to enhanceprotection of dopaminergic neurons in the striatum from the damageassociated with MPTP treatment.

In vitro dopaminergic neuronal cell cultures are prepared by dissectingthe midbrain floor plate from gestation day 14 Wistar rat embryos. Thetissue is dissociated with trypsin and seeded at a density of about2×10⁵ cells/cm² on polyorthinine-laminin coated glass coverslips. Thecells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days and are processed for tyrosinehydroxylase, a specific marker for dopminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time. Since the dopaminergic neuronsare isolated from animals at gestation day 14, a developmental timewhich is past the stage when the dopaminergic precursor cells areproliferating, an increase in the number of tyrosine hydroxylaseimmunopositive neurons represents an increase in the number ofdopaminergic neurons surviving in vitro.

Example 33

Supression of TNF-α induced adhesion molecule expression. Therecruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-α), is a proinflammatory cytokine andstimulates all three CAMs on endothelial cells. It may be involved in awide variety of inflammatory responses, often resulting in apathological outcome. The capacity of a formula 1 compound to mediate asuppression of TNF-α induced CAM expression can be examined. A modifiedELISA assay which uses ECs as a solid phase absorbent is employed tomeasure the amount of CAM expression on TNF-α treated ECs whenco-stimulated with a member of the FGF family of proteins. To performthe experiment, human umbilical vein endothelial cell (HUVEC) culturesare obtained from pooled cord harvests and maintained in growth medium(EGM-2, Clonetics, San Diego, Calif.) supplemented with 10% FCS and 1%penicillin/streptomycin in a 37° C. humidified incubator containing 5%CO₂. HUVECs are seeded in 96-well plates at concentrations of about1×10⁴ cells/well in EGM medium at 37° C. for 18-24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 optionally supplemented with 100 U/mLpenicillin and 100 mg/mL streptomycin, and treated with a given cytokineand/or growth 5 factor(s) for 24 h at 37° C. Following incubation, thecells are then evaluated for CAM expression.

HUVECs are grown in a standard 96 well plate to confluence. Growthmedium is removed from the cells and replaced with 90 μL of 199 Medium(10% FBS). Samples for testing and positive or negative controls areadded to the plate in triplicate (in 10 μL volumes). Plates areincubated at 37° C. for either 5 h (selectin and integrin expression) or24 h (integrin expression). Plates are aspirated to remove medium and100 μL of 0.1% paraformaldehyde-PBS (with Ca⁺⁺ and Mg⁺⁺) is added toeach well. Plates are held at 4° C. for 30 min. Fixative is then removedfrom the wells and wells are washed 1× with PBS(+Ca,Mg)+0.5% BSA anddrained. Do not allow the wells to dry. 10 μL of diluted primaryantibody is added to the test and control wells. Anti-ICAM-1-Biotin,Anti-VCAM1-Biotin and Anti-E-selectin-Biotin are used at a concentrationof 10 pg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells areincubated at 37° C. for 30 min. in a humidified environment. Wells arewashed ×3 with PBS (with Ca, Mg) and 0.5% BSA. Then add 20 μL of dilutedExtrAvidin-Alkaline Phosphotase (1:5,000 dilution) to each well andincubate at 37° C. for 30 min. Wells are washed ×3 with PBS (with Ca,Mg) and 0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolvedin 5 mL of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycinebuffer is added to each test well. Standard wells in triplicate areprepared from the working dilution of the ExtrAvidin-AlkalinePhosphotase in glycine buffer: 5 μL of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then be added toeach of the standard wells. The plate is incubated at 37° C. for 4 h. Avolume of 50 μL of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well. Results are indicated as amount of bound APconjugate ineach sample.

Example 34

Inhibition of a mixed lymphocyte reaction. This assay can be used toevaluate inhibition of a Mixed Lymphocyte Reaction (MLR) by formula 1compounds. Inhibition of a MLR may be due to a direct effect on cellproliferation and viability, modulation of costimulatory molecules oninteracting cells, modulation of adhesiveness between lymphocytes andaccessory cells, or modulation of cytokine production by accessorycells. Multiple cells may be targeted by the compounds since theperipheral blood mononuclear fraction used in this assay includes T, Band natural killer lymphocytes, as well as monocytes and dendriticcells. Compounds that inhibit the MLR are useful in treating, preventingor ameliorating diseases associated with lymphocyte and monocyteactivation or proliferation. These include, e.g., inflammatory orautoimmune conditions such as asthma, arthritis, diabetes, inflammatoryskin conditions, psoriasis, eczema, systemic lupus erythematosus,multiple sclerosis, glomerulonephritis, inflammatory bowel disease,Crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graftvs. host disease, host vs. graft disease and hepatitis.

To perform the assay, PBMCs from, e.g., human, donors are purified bydensity gradient centrifugation using Lymphocyte Separation Medium(LSM™, density 1.0770 g/mL, Organon Teknika Corporation, West Chester,Pa.). PBMCs from two donors are adjusted to 2×10⁶ cells/mL in RPMI-1640(Life Technologies, Grand Island, N.Y.) supplemented with 10% FCS and 2mM glutamine. PBMCs from a third donor is adjusted to 2×10⁵ cells/ml.Fifty μL of PBMCs from each donor is added to wells of a 96-well roundbottom microtiter plate. Several concentrations of the formula 1compound in the wells is used in triplicate, e.g., 1 nm, 10 nm, 100 nm,1 μm and 10 μm. Recombinant human IL-2 (R&D Systems, Minneapolis, Minn.,catalog number 202-IL) is added to a final concentration of about 0.1 to1 μg/mL and anti-CD4 monoclonal antibody (e.g., R&D Systems, clone34930.11, catalog number MAB379) is added to a final concentration ofabout 1 to 10 μg/mL. Cells are cultured for 7-8 days at 37° C. in 5%CO₂, and 1 μCi of [³H] thymidine is added to wells for the last 16 hrsof culture. Cells are harvested and thynudine incorporation determinedusing a suitable scintillation counter, e.g., a Packard TopCount. Datais expressed as the mean and standard deviation of triplicatedeterminations. Samples of the protein of interest are screened inseparate experiments and compared to the negative control treatment,anti-CD4 mAb, which inhibits proliferation of lymphocytes and thepositive control treatment, IL-2 (either as recombinant material orsupernatant), which enhances proliferation of lymphocytes.

Previously described MLR protocols, sources of cells for use in the MLRand assay parameters can be used to evaluate the effects of the formula1 compounds. See, e.g., K. V. Bromelow et al., J. Immunol. Methods 2001247:1-8, Z. Amirghofran et al., J. Ethnopharmacology 2000 721:167-172,T. Itoh et al., J. Antibiot (Tokyo) 1993 46:1575-1581, P. Van Vlasselaeret al., Cell. Immunol. 1991 138:326-340, and A. Shaked et al.,Transplantation 1991 52:1068-1072.

Example 35

Effects on the CNS. The effects of the formula 1 compounds on memory,learning, motor function or the status of a neurological condition orneurodegeneration condition are assayed using standard methods. Forexample, aged, two year old mice are tested in the Morris water mazeprocedure by training the mice to locate a pedestal in less than 15seconds in three consecutive trials. Immediately upon completion oftraining one group of mice is treated with a formula 1 compound (5-30mg/kg) and a second group is treated with a placebo. The treatmentcomprises one, two or three intraperitoneal, subcutaneous, intramuscularor intravenous injections of the formula 1 compound and the vehicleplacebo. The injections are given once per day. Two weeks aftertreatment, the time to rescue is timed in the Morris water mazeprocedure and the control result is compared to the placebo control. Theuse of Morris water maze and other procedures to measure the effect ofvarious conditions or compounds on learning, memory or neurologicalconditions have been described, see e.g., R. Gerlai Trends Neurosci.1996, 19:177-181, J. L. W. Lau et al., Proc. Nat'l. Acad. Sci. 2001,98:4716-4721, U.S. Pat. Nos. 6,348,489, 6,251,942 and 6,277,886.

Scopolamine induced amnesia is examined essentially as follows. Groupsof 13 to 16 C57BL76 mice (about 35 gm) are trained in the Morris watermaze procedure to locate a pedestal in less than 15 seconds in threeconsecutive trials. Immediately upon completion of training the mice ineach of three groups are treated with scopolamine (1 mg/kg), scopolamineplus a formula 1 compound at one or more dosages (e.g., about 5-50mg/kg), and scopolamine plus a placebo. The treatment comprises one, twoor three intraperitoneal, subcutaneous, intramuscular or intravenousinjections of the formula 1 compound and the vehicle placebo. Theinjections are given once per day. Six days after treatment the averagetime (sec) to rescue is timed using the Morris water maze procedure andthe results from each group are compared. Results for a formula 1compound such as 16α-fluoroandrost-5-ene-17-one or7-hydroxy-16α-fluoroandrost-5-ene-17-one are optionally compared to theresults that are obtained in these protocols using another controlcompound, e.g., (S)-(−)—N-propargyl-1-aminoindan or nefiracetam, oranother formula 1 compound such as BrEA or AET to determine the relativepotency of the formula 1 compounds with respect to each other.

Example 36

The capacity of formula 1 compounds to limit injury associated withischemia and reperfusion is determined in an animal model essentially asfollows. Male Sprague-Dawley rats weighing 130-170 g are randomlyassigned to no pre-treatment, vehicle pre-treatment or formula 1compound pre-treatment using one or more dosages, e.g., about 1-10mg/kg. Animals are treated with vehicle or DHEA the day before and theday of surgery. Anesthesia is induced with intraperitoneal pentobarbital(60-70 mg/kg). The rats are placed on a heating pad, and bodytemperature is maintained at about 36° C. Detection of the cremastermuscle on its neurovascular pedicle is performed essentially accordingto conventional techniques, e.g., Anderson, G. L. et al., MicrovascularRes. 1988 36:56-63, Siemionow, M. et al., Microcirc. Endoth. Lymphatics1991 7:183-197, Siemionow, M. et al., J. Hand Surgery 1993 18A:963-971.

Briefly, a skin incision is made from the anterior iliac spine to thetip of the scrotum. The testis with cremaster muscle intact is thendissected away from the scrotum. An opening of 1 cm is made on theventral surface of the cremaster, and the testis and spermatic cord areremoved. Under a microscope, the neurovascular pedicle, consisting ofthe pubic-epigastric arteries, vein, and genitofemoral nerve, is thencompletely isolated by dissecting to the origin of the vessels from theexternal iliac artery and vein. The front wall of the cremaster musclesac is opened and the island cremaster muscle flap is prepared forintravital videomicroscopy. The rat is secured on a tissue bath, and thecremaster muscle flap is spread over the coverglass in the opening atthe bottom of the bath and fixed with 5-0 silk sutures. It is thentransilluminated from below, using a fiber optic tungsten lamp. Themuscle is kept moist and covered with impermeable plastic film. Thetissue bath, designed specifically for temperature control, is filledwith 0.9% saline and the temperature maintained at between 35-36° C. Themicroscope is equipped with a color video camera. The video image of themicrocirculation is displayed on a 19″ monitor, where the finalmagnification is 1800×. Measurement of microvascular activity isrecorded after isolation of the muscle to establish the pre-ischemiabaseline. After proper positioning of clamps to completely shut downblood flow to the muscle flap, the duration of the ischemic period issix hours. Following removal of clamps to induce reperfusion injury,activity in the microvasculature is measured at e.g., 30, 60 and 90minutes post-reperfusion. In all experimental subjects, ischemia isfollowed by reflow and then by an initial period of flow of bloodthrough the microcirculation. This burst of circulatory activity isfollowed by marked reperfusion injury that induces loss of flow.

One or more of the following parameters are used to evaluate the stateof the cremaster muscle microvasculatory system prior to ischemia andafter reperfusion. The density of perfused capillaries in each of threeflap regions is measured by counting the number of flowing capillariesin proximity to the preselected post-capillary venule. Nine visualfields of capillaries are counted at each postcapillary venule site, fora total of 27 fields per cremaster muscle flap.

A leukocyte count in postcapillary venules is taken using video scans ofthree pre-selected post-capillary venules in proximal, middle and distalflap regions. For each venule, the number of leukocytes rolling throughthe lumen, the number adhering to the endothelium and the numbermigrating across the endothelium over a two-minute period are recorded.Results are optionally obtained for rollers, strikers and diapedesis.

Red blood cell velocities in first order and second order arterioles aremeasured. Red blood cell velocities are recorded in the main arteriolesof the cremaster flap using an optical Doppler velocimeter. Results areobtained for velocity of venous and arterial blood.

In an exemplary protocol, six rats are untreated and six rats arepre-treated with vehicle. Under conditions of six hours of ischemia and90 minutes of reperfusion, the absolute number of rolling, sticking andtransmigrated leukocytes is determined within 60 minutes of reperfusionand at 90 minutes. Rats are pre-treated with a formula 1 compound bysubcutaneous injection the day before and the day of surgery to measureany protective effect of the therapy. One or more of the threeparameters are determined and are compared to normal values. Theendothelial-adherent properties compared to baseline values areoptionally determined, using numbers of rolling, sticking andtransmigrating leukocytes. Red cell velocities in second orderarterioles are compared to normal rates of flow at, e.g., 90 minutespost-reperfusion.

Example 37

Pulmonary vasoconstriction. The capacity of formula 1 compounds to limithypoxia induced pulmonary vasoconstriction is demonstrated using ananimal model essentially as follows. Isolated perfused ferret lungs arean established animal model to study secondary pulmonary hypertension.In brief, male ferrets are anesthetized with intraperitonealpentobarbital sodium and the chest is opened. Stainless steel cannulaeare secured in the left atrium and pulmonary artery, and the pulmonaryartery and the aorta are ligated. The lungs are perfused with a mixtureof autologous blood and Krebs-Henseleit buffer in a circulating mannerat a constant rate of about 85 mL/min. The perfusion circuit includes aperfusate reservoir, a roller perfusion pump, filter, and a heatexchanger. The perfusion system is made of, e.g., tygon tubing, which isused for connections and for passage through the perfusion pump. Thetemperature of the perfusate is kept about 37-38° C. and the pH ismaintained at 7.35 to 7.40 by adding sodium bicarbonate to the reservoiras needed. The venous reservoir is placed below the lowermost portion ofthe lung.

The lungs are ventilated with a hypoxic gas mixture of 5% CO₂, 4% O₂,and 91% N₂ by a tracheotomy with a Harvard animal respirator for 30minutes. The animals are ventilated with a tidal volume of 30 mL, at arate of 18 breaths/min. and with 2 cm of H₂O positive end-expiatorypressure. For measurements, pulmonary arterial, left atrial and trachealpressures are monitored using Gould Statha P231 D pressure transducersor an equivalent connected to the inflow circulation and recorded on,e.g., a Grass polygraph. After 30 minutes of ventilation with hypoxicgas mixture, a formula 1 compound in a dose between about 5-25 mg/kgbody weight is added to the reservoir, and perfusate is allowed toperfuse the ferret lungs for 1.5 hours. Pulmonary artery pressure ismeasured until the end of the experiment, i.e., a total of two hours.Pressure that remains at or near basal level indicates the vasodilatoryeffect of the formula 1 compound in pulmonary circulation that isotherwise constricted in response to hypoxia. The effects of the formula1 compounds can be compared to the effects and duration of nitric oxide,a therapeutic agent that is optionally used in this model as a control.

Example 38

Hemopoiesis modulaton. Enhanced hemopoiesis is observed in mammals withimmune injury from, e.g., radiation exposure or from animmunosuppressive chemotherapy. In an example, animals are used todemonstrate the effect of formula 1 compounds on hemopoiesis afterimmune system injury due to radiation. Hemopoiesis in the murine immunesystem after radiation is optionally used because of the similarresponses of murine and human hemopoiesis to drugs and toxic insults(see, e.g., J. H. Hendry and B. I. Lord, editors, Radiation toxicology:Bone marrow and leukaemia 1995 Taylor & Francis Inc., London).

In an exemplary protocol, B6D2F1/J female mice (Jackson Laboratory, BarHarbor, Me.), 18-24 weeks of age, 22-30 g body weight, are obtained andheld in quarantine for two weeks. Up to 10 mice are housed in sanitized46×24×15 cm polycarbonate boxes with filter covers (MicroIsolator; LabProducts, Inc, Maywood, N.J.) on autoclaved hardwood chip bedding. Miceare given feed and acidified (pH 2.5) water freely. The animal holdingroom is maintained with conditioned fresh air at approximately 21° C.and 50° (±10%) relative humidity and with a 12-h light/dark fullspectrum lighting cycle.

Mice are placed in ventilated Plexiglas containers and exposedbilaterally to gamma-radiation from a ⁶⁰Co source. Exposure time isadjusted so that each animal received a midline tissue-absorbed dose of1-12 Gy at a nominal dose rate of 0.4 Gy/min at ambient temperature.Using a standardized technique, the midline dose rate is measured byplacing a 0.5 cc tissue-equivalent ionization chamber at the center of a2.5-cm diameter cylindrical acrylic mouse phantom. The tissue-air ratio,defined as the ratio of the dose rate measured in the phantom to thedose rate in free air, for this array is about 0.96. Variation withinthe exposure field is less than about 4%. Dosimetric measurements aremade in accordance with the American Association of Physicists inMedicine protocol for the determination of absorbed dose fromhigh-energy photon and electron beams (Med. Phys. 1983 10:741-771).Sham-irradiated mice are treated in the same way as the irradiatedanimals, except that the animals are not irridiated.

Various formula 1 compounds e.g., compounds such as those in thecompound groups described herein are formulated with a suitable vehicle(e.g., PEG-400) or sterile 0.9% NaCl (saline) optionally containingother excipients such as a cyclodextrin. The compounds are injectedsubcutaneously in a volume of about 0.1 mL or they are delivered orallyor they are administered by another route. Doses typically range fromabout 1 mg/kg to about 350 mg/kg, e.g., about 1, 10, 20, 40, 80, 160 or320 mg/kg.

Blood (0.6-1.0 mL) is obtained from halothane-anesthetized mice bycardiac puncture using a heparinized syringe attached to a 21-gaugeneedle. Blood is collected in EDTA-containing sample tubes. Mice areeuthanized by cervical dislocation after blood collection. White bloodcell (WBC), red blood cell (RBC) and platelet (PLT) counts are performedusing, e.g., a Hematology System 9000 (Biochem Immunosystems).Wright-stained blood smears from the same samples are made fordifferential counts of neutrophils and lymphocytes by light microscopy.

Hemopoietic progenitor cells committed to granulocyte-macrophagedifferentiation (GM-CFC) are assayed by a single-layer modification of adouble-layer semisolid agar technique essentially as described (Patchenet al. Adv. Space Res. 1992 12:223-248). For example, femoral marrow isextracted and cell suspensions are prepared by flushing with 3 mL ofMcCoy's 5A medium containing 10% heat-inactivated fetal bovine serum(HIFBS; Hyclone, Logan, Utah). Each cell suspension represented a poolof marrow from four femurs, i.e., both femurs from each of two mice. Thetotal number of nucleated cells in each suspension is determined with,e.g., a Coulter counter. The agar-medium mixture consisted of equalvolumes of 0.66% agar and double-strength supplemented CMRL 1066 medium(Gibco, Grand Island, N.Y.). The medium is supplemented with finalconcentrations of 10% HIFBS, 5% tryptic soy broth, 5% heat-inactivatedhorse serum, antibiotics, and L-serine. One milliliter of theagar-medium mixture is added to each 35-mm plastic Petri dish (twodishes per suspension) and mixed with 50 μL of 0.1 ng/μL recombinantmouse GM-CSF (Genzyme, Cambridge, Mass.). Cell suspensions are thenmixed into the agar-medium mixture to a final concentration of 0.5×10⁵cells/mL for unirradiated animals, and 1.0×10⁵ or 1.5×10⁵ cells/mL forirradiated animals to ensure sufficient colonies per plate forquantitation. Control experiments are done to confirm linearity ofcolonies at cell concentrations of 0.5−1.5×10⁵ cells/mL. Colonies (>50cells) are counted after seven days incubation in a 37° C. humidifiedenvironment containing 5% CO₂. The average of the counts for the twodishes is taken as the value for each pool. About six animals are usedper group in each of two experiments.

For histological examination of myeloid hyperplasia in bone marrow afteradministration of the formula 1 compound, mice are euthanized withhalothane, tissues are immersed in formalin, bones are decalcified androutine H&E-stained 6-μm paraffin sections are prepared.

For induced-infection studies, a clinical isolate of K. pneumoniae,capsule type 5 (strain AFRRI 7), that is kept frozen at 70° C. in skimmilk, is grown overnight at 35° C. on Trypticase Soy Agar(Becton-Dickinson, Sparks, Md.). Five typical colonies are inoculatedinto 8 mL of brain heart infusion broth (Becton-Dickinson) and incubatedovernight at 35° C. Two milliliters of this overnight suspension isinoculated into 95 mL of prewarmed brain heart infusion broth. Theculture is incubated at 35° C. with shaking for approximately 2.5 h. Theoptical density of bacterial growth is monitored with aspectrophotometer at a wavelength of 550 nm. Late log-phase cells areished and suspended in cold saline to yield 10⁹ viable bacteria per mL.Appropriate dilutions for inoculations are made in cold saline.

To induce a bacterial infection, all mice are injected sc with K.pneumoniae four days after sham-irradiation or irradiation whencirculating leukocytes are depressed. Mice are inoculated sc rather thaniv or ip, to establish infection leading to sepsis, but not rapid septicshock. After sc inoculations of K. pneumoniae in the mice, the infectionremains largely localized to the injection site. K. pneumoniae are notdetectable in blood of inoculated mice until a few hours before death.

Different doses of the bacteria are inoculated for each of threeradiation dose levels (0, 1 or 3 Gy) to approximate the LD_(95/30),because the effects of radiation on hemopoiesis and susceptibility toinfection are dependent on the dose of radiation. The LD_(95/30) forbacteria at each radiation dose is calculated from probit analysis. Theactual doses are estimated by dilution plating of inocula ontoTrypticase Soy Agar and incubating overnight at 35° C. Since differentbacterial doses are expected to be needed for different radiation doses,the LD_(95/30) is estimated for each group and different mortality ratesare observed in the vehicle-injected control groups. Bacterial doses forinduced-infection experiments are prepared and calculated in the samemanner.

Animals are checked frequently, e.g., once or twice daily, six or sevendays per week, to monitor survival and to euthanize mice that are in amoribund state. To verify that mortality in the induced-infectionexperiments is associated with K. pneumoniae injection, heart blood fromrecently deceased animals (or moribund animals euthanized by cervicaldislocation) is cultured overnight at 35° C. on Columbia sheep bloodagar plates (Becton-Dickinson, Sparks, Md.). Colonies are identified asK. pneumoniae by a suitable means, e.g., Biolog analysis.

For histological analysis of bone marrow, coded slides are scored blindusing a five-level semiquantitative scale and the results analyzed witha randomization t-test to obtain exact P-values. Thirty-day survivalvalues are compared using the generalized Savage (Mantel-Cox) procedure(BMDP Statistical Software, Inc, Los Angeles, Calif.). To calculate dosereduction factors (DRFs), probit analysis is performed on mortalitydata.

To test the ability of formula 1 compounds to ameliorateradiation-induced defects in hemopoiesis, mice are exposed to bilateralwhole-body gamma-radiation and receive a dose of 3 Gy (or aresham-irradiated). One hour after irradiation or sham-ir-radiation, miceare injected with 320 mg/kg 3β,17β-dihydroxyandrost-5-ene (“AED”) orPEG-400 vehicle. Between-group differences in blood cell elements, e.g.,neutrophils, GM-CFC and platelets are generally determined. Irradiationresults in a decrease in neutrophils at about four days after radiationcompared to sham-irradiated animals.

Example 39

BrEA was administered to relatively late stage HIV-infected patients(CD4 counts of ≦100 cells/mm³, HIV RNA at screening of ≦1×10⁶ copies/mL)at least 18 years old who were at risk of developing opportunisticinfections, but who had adequate hematological and renal function, e.g.,WBC counts of ≧1.0×10⁹ cells/L, absolute neutrophil counts of ≦0.75×10⁹cells/L, hemoglobin of ≦7 g/dL and serum creatinine of 132 μmol/L. BrEAand vehicle placebo was administered by intramuscular injections of 50or 100 mg per dose in a 1 or 2 mL volume. The treatment protocol isongoing.

The incidence rate, time to resolution and time to recurrence ofopportunistic infections is determined for patients receiving BrEA andfor control patients receiving placebo during treatment and optionallyevery month for 3 or 4 months following the last treatment course. Atreatment course consisted of intramuscular injections given daily for 5consecutive days and repeated every 6 weeks. Treatment course oneinjections started on day 0. Treatments are optionally carried out for atotal of 7 courses over 42 weeks.

In patients examined at day 4, 43, 46 and 56 after 1 course oftreatment, the level of RNA for several inflammation associated geneswas analyzed. The RNA was obtained from circulating white blood cellsfrom the patients and the uninfected volunteers. Peripheral blood wascollected into a CPT-Vacutainer (Becton Dickinson) and PBMC isolationwas performed according to the manufacturer's protocol. The PBMC weremaintained in 1 mL of RPMI 1640 with 10% FCS at 37° C. for 3 hours andthen lysed in lysis buffer (300 μL MagnaPure™). RNA levels from PBMClysates were measured by preparation of cDNA using commercial AMVreverse transcriptase and PCR kits and protocols (First Strand cDNASynthesis™, Roche Diagnostics; LightCycler FastStart DNA Sybr Green I™Kit, Roche Diagnostics; LightCycler Primer sets, Search-LC, Heidelberg).The results generally showed a detectable decrease compared to baselinelevels of about 40-98%, generally about 50-90% for RNA encoding IL-1β,TNFα, IL-6, IL-8, IL-10, COX-2 and MCP-1. The level of GM-CSF wasincreased at 43 days and decreased at all of the other time points. Inthe HIV-infected patients before treatment with BrEA, compared tohealthy uninfected, i.e., not HIV infected, volunteers, there was astatistically significant (Mann-Whitney analysis) increase in RNAencoding IL-1β, TNFα, MIP-1α, IL-6, IL-8, COX-2, M-CSF, GM-CSF, MCP-1and IFNγ. BrEA treatment for 5 days thus resulted in a decrease inmultiple inflammation-associated markers.

Other details of similar clinical protocols may include at least some ofthe following procedures. Prior to randomization, patients areoptionally screened for acute, active HIV related infections oropportunistic infections. Screening may include vital sign measurements(temperature, heart rate, sitting blood pressure, and oxygen saturationby pulse oximetry), a chest X-ray, a complete physical examinationincluding ophthalmic exam with funduscopy and pelvic examinationincluding a PAP smear, and a complete blood laboratory examination.Additionally, patients may provide sputum samples to identify active TBinfection and stool samples for culture and sensitivity for ova andparasites. Patients who require acute treatment, hospitalization, orchemotherapy for active infections are optionally excluded fromparticipation.

The patients are optionally monitored for safety throughout the studyand, e.g., every month for 3 months following the last treatment course.Patients who discontinue due to an clinically significant opportunisticinfection are optionally followed for resolution and outcome. Patientsmay be evaluated for opportunistic infections (Ols) at the time ofdosing. Suspected opportunistic infections are usually diagnosed byappropriate diagnostic methods. Opportunistic infections are generallydocumented and their course followed throughout the study. Theopportunistic infections may be evaluated as protocol endpoints and mayinclude one or more of tuberculosis, candidiasis, PCP, diarrhea, or anyopportunistic infections such as Cryptosporidiosis (e.g., with >1 monthdiarrhea), Isosporiasis (e.g., with >1 month diarrhea), Microsporidiosis(e.g., with >1 week diarrhea or other manifestations), Pneumocystiscarinii (pneumonia or extrapulmonary infection), Toxoplasmosis, Kaposi'ssarcoma, invasive cervical cancer, cervical dysplasia, Candidiasis(oropharyngeal, vaginal, esophageal or pulmonary), Coccidiomycosis(disseminated or extrapulmonary), Aspergillosis (invasive ordisseminated), CMV infection, an active Herpes simplex infection(bronchitis, pneumonitis, or esophagitis), progressive multifocalleukoencephalopathy, systemic Mycobacterium avium complex infection or anon-typhoidal Salmonella septicemia.

Microsporidiosis (with >1 month diarrhea or other manifestations)requiring premature study discontinuation. Patients may be prematurelydiscontinued from the study if they experience a serious opportunisticinfection that requires more than ten days of hospitalization, or if theopportunistic infection does not respond to a specific treatment aftertwo weeks of acute primary and/or secondary treatment. Patients whodiscontinue the study due to a serious opportunistic infection shouldcomplete the premature discontinuation visit, if possible. Whenever apatient is diagnosed with one or more opportunistic infections, anappropriate treatment is typically started based on the normal standardof care and/or the caregiver's judgment. Eligible patients areoptionally provided prophylaxis treatment at the baseline visit for PCP.In addition, patients are optionally provided with a nutritionalsupplement of multivitamins with minerals.

All patients are optionally monitored for one or more of HIV RNA levels(e.g., by Chiron Quantiplex™ branched chain DNA assay), T-cell subsets(CD4/CD8), plasma dehydroepiandrosterone sulfate, plasma cortisol.Immunological analysis may include cytoplasmic or serum cytokines orinterleukins (e.g., IL-2, 4, 6, 8, 10, and 12), gamma interferon,insulin-like growth factor (IGF-1) and tumor necrosis factor (TNFα),RANTES and expression of the protein or messenger RNA products for theseor other genes.

Assessment of the patient's immune status by performing delayedhypersensitivity (DTH) skin testing may be done and read at 48 and/or 72hours after application but prior to administering BrEA. The skin testis optionally done at the baseline visit, usually within two weeks ofthe initial dosing of BrEA. Skin test antigens for DTH may be repeatedat later times, e.g., at weeks 6, 10, 18, 30, 42 and 57.

At clinic visits various examinations, including a thorough pulmonaryexam may be done. This includes but is not limited to measuring bodyweight, Karnofsky Performance status, determining oxygen saturation andvital signs. Patients may be queried at visits with the caregiver foradverse events, symptoms of a suspected or existing opportunisticinfection as well as any new opportunistic infection, HIV related eventsand changes in drug therapy.

Patients are dosed at the following times for the 5-day course ofintramuscular injections: Week 1 (days 0-4); week 7 (days 42-46); week13 (days 84-88); week 19 (days 126-130); week 25 (days 168-172); week 31(days 210-214) and week 37 (days 252-256) for a total of seven treatmentcourses. BrEA or placebo is supplied as a liquid formulation in aliquotsin 2 mL amber glass vials. Each vial with BrEA contains 50 mg of BrEAper mL in a vehicle formulation consisting of 40% polyethylene glycol200, 2% benzyl benzoate, 2% benzyl alcohol and qs propylene glycol. BrEAshould be injected slowly into the muscle. Pregnant females areoptionally excluded from treatment. The use of some other compounds areoptionally prohibited during the study (e.g., cancer chemotherapy,Moducare, testosterone, DHEA, deca-durabolin or oxandrolone within,e.g., 4 weeks of study initiation, or hydroxyurea, methotrexate,antiretroviral agent treatments).

Example 40

BrEA was administered to treatment naïve HIV-infected patients. Thesepatients had a total of 2 weeks or less of any prior antiretroviraltreatment. The patients had CD4 counts of ≧200 cells/mm³, HIV RNA atscreening between 5,000 and 1×10⁶ copies/mL. They were at least 18 yearsold and had adequate hematological and renal function, e.g., WBC countsof ≧1.0×10⁹ cells/L, absolute neutrophil counts of ≧0.75×10⁹ cells/L,hemoglobin of ≧7 g/dL and serum creatinine of <132 μmol/L. BrEA wasadministered by subcutaneous injection of 50 or 100 mg per dose usingthe formulation described in the pervious example. The treatmentprotocol is ongoing.

After or during one treatment course of 100 mg of BrEA for 5 consecutivedays, the HIV infected patients were examined at days 2-35. Day 1 wasthe first treatment day. The level of RNA for several inflammationassociated genes was analyzed. At day 5, the results showedstatistically significant decrease compared to baseline levels of RNAencoding IL-1β, TNFα, GM-CSF, MIP-1α and COX-2, and a statisticallysignificant increase in PPARγ. The RNA was obtained from circulatingwhite blood cells from the patients and the uninfected volunteersessentially as described in the preceeding example. In the HIV-infectedpatients before treatment with BrEA, compared to healthy uninfected,i.e., not HIV infected, volunteers, there was a statisticallysignificant (Mann-Whitney analysis) increase in RNA encoding IL-1β,TNFα, MIP-1α, IL-6, IL-8, IL-10, COX-2, M-CSF, RANTES, GM-CSF, MCP-1 andIFNγ. At days 2-35 a statistically significant decrease (p<0.001 for allmarkers) in IL-1β, IL-6, TNFα, GM-CSF, MIP-1α, MCP-1 and COX-2transcripts was observed and an increase in PPARγ (p=0.034) wasobserved. Increases in PPARα and t-Bet was also observed in the treatedpatients.

BrEA treatment for 2-5 days thus resulted in a modulation of multipleinflammation-associated markers, i.e., decreased proinflammatorycytokines and increased antiinflammatory markers.

Other details of similar clinical protocols may include at least some ofthe following procedures. The incidence rate, time to resolution andtime to recurrence of toxicities or opportunistic infections isdetermined for patients receiving BrEA during the treatment andoptionally every month for 3 or 4 months following the last treatmentcourse. BrEA and the placebo vehicle are administered as a 1 mLsubcutaneous injection. A treatment course consists of subcutaneousinjections given daily for 5 consecutive days and repeated every 4, 5 or6 weeks for a total of 3 or 4 courses over about 12-24 weeks. Treatmentcourse one injections start on day 0. About 24 patients are randomizedto different treatment groups: BrEA 50 mg or placebo equivalent or BrEA100 mg or placebo equivalent. In each treatment group, patients areoptionally randomized in a 2:1 or a 3:1 ratio to either active drug, 50or 100 mg BrEA, or the placebo.

The day each patient receives a first of five once daily doses is day 1.In one treatment protocol, the 5-day treatment course is followed by a 5week observation period and the patients receive a total of 3 treatmentcourses consisting of 5 consecutive days of once daily dosing of BrEA orplacebo equivalent followed by a 5 week observation period. Patientsreceive 4 treatment courses over a 24-week period. In this protocolApproximately 24 patients will be randomized to one of two treatmentgroups. Within each treatment group, patients are randomized in a 3:1ratio to BrEA or placebo equivalent (9 patients randomized to BrEA and 3patients randomized to placebo equivalent).

Pregnant females are optionally excluded from treatment. The use of someother compounds are optionally prohibited during the study (e.g., cancerchemotherapy, Moducare, testosterone, DHEA, deca-durabolin oroxandrolone within, e.g., 4 weeks of study initiation, or hydroxyurea,methotrexate, antiretroviral agent treatments).

All patients are optionally monitored for levels of HIV RNA (ChironQuantiplex™ branched chain DNA assay) and T-cell subsets (CD4/CD8).Activation markers/immunological analysis may include, but are notlimited to, cytoplasmic cytokines, serum cytokines, gene expression,interleukins (IL-2, 4, 6, 8, 10 and 12), gamma interferon, insulin-likegrowth factor and tumor necrosis factor alpha. Additional researchtesting may be conducted on samples collected at each visit. Chemistryand hematology panels and urinalysis will be performed as indicated inthe schedule of evaluations. Additionally, patients co-infected withhepatitis B and/or C viruses, malaria or tuberculosis are optionallymonitored regularly for viral titers or microbiological cultures.

Example 41

BrEA was administered by transmucosal (buccal) administrations of BrEAto HIV-infected patients who were treatment-naïve (no previous treatmentor less than 2 weeks of previous treatment with antiretroviral agents)and who had a viral load greater than 500 HIV copies/mL and a CD4 cellcount ≧200 cells/mm³. The treatment protocol is ongoing. BrEA or aplacebo equivalent is administered by transmucosal delivery routeaccording to the following 3 dosing groups.

For Group 1, a single treatment course is 50 mg of HE2000 or placeboadministered for five consecutive days over one week followed by afive-week observation period. Each treatment course is six (6) weeks induration and each patient receives three consecutive treatment coursesover a total of 18 weeks. For Group 2, a single treatment course is 50mg of BrEA or placebo given three times a week over four weeks followedby a two-week observation period. Each treatment course is six (6) weeksin duration and each patient receives three consecutive treatmentcourses over a total of 18 weeks. For Group 3, a single treatment courseconsists of 50 mg of BrEA or placebo given once a week over six weeks.Each treatment course is six weeks in duration and each patient receivesthree consecutive treatment courses over a total of 18 weeks. About 36patients are enrolled (e.g., 12 patients per dosing Group). The dosingis summarized below.

DOSING NO. OF BREA GROUP PATIENTS DOSE 1 9 50 MG 1 3 PLACEBO (0 MG) 2 950 MG 2 3 PLACEBO 3 9 50 MG 3 3 PLACEBO

The day the patient receives the first dose is day 1. A six-week periodencompasses one treatment course. The patients are optionally followedby monthly follow-up visits for 1, 2 or 3 consecutive months afterdosing.

A preliminary analysis of group 1 patients at day 5 indicated a generaldecrease in one or more inflammation-associated markers, e.g., IL-1p,TNFα, MIP-1α, COX-2 and GATA-3. In these HIV-infected patients beforetreatment with BrEA, compared to healthy uninfected, i.e., not HIVinfected, volunteers, there were statistically significant (Mann-Whitneyanalysis, p<0.001) increases in RNA encoding IL-1p, TNFα, MIP-1a, IL-6,IL-8, IL-10, COX-2, M-CSF, RANTES, GM-CSF, MCP-1 and IFNγ. BrEAtreatment in these patients was consistent with modulation ofsignificant immune markers.

BrEA is provided as transmucosal or buccal tablets. Each tabletcontained 25 mg of BrEA and sucrose, lactose, and Tween 80 excipients.Placebo transmucosal tablets containing the excipients, but lacking theBrEA are provided. Patients are instructed to drink water before placingtwo buccal tablets (50 mg dose) between the upper gingival surfaces ofthe jaw and buccal mucosa of the cheek pouch. The tablets are to be heldin the mouth without disturbance for about 10 minutes. During this time,patients are observed to ensure that the tablets are not chewed orswallowed.

Other details of similar clinical protocols may include at least some ofthe following procedures. All patients are optionally monitored forlevels of HIV RNA (Chiron Quantiplex™ branched chain DNA assay) andT-cell subsets (CD4/CD8). Immunological analyses may be conducted in asubset of patient samples and may include cytoplasmic cytokines, serumcytokines, gene expression, interleukins (IL-2, 4, 6, 8, 10 and 12),gamma interferon, insulin-like growth factor and tumor necrosis factor.Additional research testing may be conducted on samples collected ateach visit. Safety laboratories, including chemistry, hematology andurinalysis are optionally performed. Patients co-infected with hepatitisB virus, C virus and/or tuberculosis, are optionally allowed on studyand are optionally monitored for viral loads and/or microbiologicalcultures.

Example 42

A buccal formulation containing BrEA was prepared and contained 1.000 kgBrEA, 3.25 Kg Fast Flo Lactose (Foremost), 0.250 kg Polyplasdone XL 10™(crospovidone NF), 0.100 kg Syloid 244FP (colloidal silicon dioxide),0.250 Kg mannitol (USP) 0.050 kg Cab-O—Sil™ (amorphous silica) and 0.100Kg magnesium stearate. Tablets containing 25 mg each of BrEA wereprepared from the mixture. Similar formulations containing other formula1 compounds, e.g., AED, AET or 7-oxodehydroepiandrosterone may containone or more of these excipients or other excipients disclosed herein.Other formulations may utilize micronized compounds or analogousexcipients, e.g., micronized BrEA hemihydrate.

Example 43

Formulations comprising formula 1 compounds were prepared and comprisedthe excipients described below. Formulations containing BrEA aregenerally stored in the dark and/or are dispensed into opaque or amberglass containers.

A liquid formulation for parenteral delivery, e.g., subcutaneous orintramuscular injection, was prepared that comprised 100 mg/mL BrEA, 2%benzyl alcohol, 30% benzyl benzoate, 30% polyethylene glycol 300 and 30%propylene glycol.

A liquid formulation for parenteral delivery, e.g., subcutaneous orintramuscular injection, was prepared that comprised 100 mg/mL BrEA, 2%benzyl alcohol, 2.5% ethanol, 50% PEG200 and qs propyleme glycol.Solubilization of the BrEA was facilitated by sonication and heating at40° C. for 5 minutes.

A liquid formulation for parenteral delivery, e.g., subcutaneous orintramuscular injection, was prepared that comprised 50 mg/mL AET, 40%PEG200, 2% benzyl alcohol, 2% benzyl benzoate and qs propylene glycol.

A liquid formulation for parenteral delivery, e.g., subcutaneous orintramuscular injection, was prepared that comprised 15 mg/mL BrEA, 45%2-hydroxypropyl-β-cyclodextrin and 0.9% saline.

A 125 mg tablet for oral or transmucosal delivery, e.g., buccal orsublingual administration, was prepared that comprised per tablet 20%w/w BrEA, 55% w/w lactose, 15% w/w mannitol, 5% w/w crospovidone, 2% w/wmagnesium stearate, 3% w/w silica.

A sterile liquid suspension formulation for intravenous delivery of aformula 1 compound is prepared that comprises about 5-60 mg/mL of aformula 1 compound such as BrEA having an average particle size of about100 to about 400 nm in saline and optionally comprising dextrose and/ora preservative such as EDTA, BHA or BHT, e.g., at about 0.1-2% w/w.

A liquid suspension formulation for transmucosal delivery, e.g., buccalor sublingual administration, was prepared that comprised 100 mg/mL ofBrEA and equal volumes of PEG300 and propylene glycol.

Example 44

A clinical trial is conducted using human patients having an acuteinfection or suspected of having been exposed to an acute infectiouspathogen. The patients are dosed with about 1 mg/kg, about 2 mg/kg,about 5 mg/kg or about 20 mg/kg of a formula 1 compound such as BrEA,3β,17β-dihydroxyandrost-5-ene or 3β,7β,17β-trihydroxyandrost-5-eneorally, by a parenteral route, e.g., by subcutaneous or intramuscularinjection, or by a transmucosal route such as by buccal, sublingual orrectal delivery. Dosages are single unit doses or subdivided andcomprise about 25 mg, about 50 mg, about 100 mg, about 200 mg or about500 mg for adults. For pediatric patients, the dosages are about 10 mg,about 20 mg, about 50 mg, about 100 mg or about 250 mg. Dosing isinitiated, e.g., at the time the patient is diagnosed as having theinfection or as having been potentially exposed to the pathogen. Thedosages are administered daily or on an intermittent basis, e.g., dailyfor about 1, 2, 3, 4, 5, 6, 7 or 8 weeks, or 2 or 3 times per week everyother day or every three days, for about 1, 2, 3, 4, 5, 6, 7 or 8 weeks,or essentially as disclosed elsewhere herein. The known or suspectedpathogen can be cells, spores or virions of Bacillus anthracis,Coccidoides immitis, Ebola virus, Lassa virus, an Aspergillus sp. fungusor a Mucor sp. fungus, an Orthopoxvirus such as the variola virus(smallpox virus) or a gram negative bacterium such as Pseudomonasaureginosa, Escherichia coli, Yersinia pestis, Vibrio cholerae orSalmonella typhi.

The patients are also generally treated with one or more antiviral,antifungal or antibacterial agents, such as one or more of thosedisclosed herein, according to normal clinical practice. Such agents areadministered before, during and/or after treatment with the formula 1compound and they are administered essentially using standard doses androutes of administration. Such agents can optionally include one, two ormore of amphotericin B, fluconazole, clotrimazole, itraconazole,ketoconazole, isoniazid, a fluororquinolone, e.g., ciprofloxacin, apenicillin, e.g., penicillin G, streptomycin, trimethoprim,tetracycline, doxycycline and erythromycin. Responses of the patientsare optionally monitored, e.g., detectable amelioration of one or moresymptoms, detectable reduction in the progression of the infection or adetectable change in an immune marker, such as one described herein,e.g., enhanced levels of molecules or cells associated with Th1 immuneresponses, a reduced level of a molecule(s) associated with inflammationsuch as TNFα, IL-1β, or IL-6, or enhanced humoral or innate immunereactions. Results obtained with patients in different groups arecompared to determine the efficacy of a formula 1 compound under theclinical protocol. For example, patients who received standard therapyand a suitable placebo are compared with results from patients whoreceived a standard therapy and a formula 1 compound.

Example 45

Enhancement of pathogen sensitivity to antimicrobial agents usingformula 1 compounds. The capacity of the formula 1 compounds to increasethe sensitivity of a bacterial, viral, fungal or yeast pathogen to aknown or candidate antimicrobial agent is examined using standardantimicrobial susceptibility assays. The assays are performed usingknown or candidate antimicrobials alone, a formula 1 compound alone, aknown or candidate antimicrobial with a formula 1 compound and asuitable untreated control(s). The antimicrobials and the formula 1compounds are used over a range of concentrations, e.g., about 0.001μg/mL to about 400 μg/mL and IC50, TC50 and/or MIC (the minimumconcentration that completely inhibits detectable growth of the pathogenor detectable cytopathic effects of the pathogen on infected cells inthe assay) values can be obtained for the antimicrobial agent usinginhibition curves. The assays are performed using in vitro assays or invivo assays, such as animal assays with, e.g., a rodent or canine.Exemplary antimicrobial and formula 1 compound concentrations include0.1 μg/mL, 0.2 μg/mL, 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20μg/mL, 50 μg/mL and 100 μg/mL. The capacity of the formula 1 compoundsto enhance the susceptibility of pathogens that are sensitive orresistant to standard or experimental therapies is examined by comparingthe efficacy of a known or candidate antimicrobial agent against thetarget pathogen in the presence and absence of a formula 1 compound andenhanced sensitivity of the pathogen in the presence of both indicates abeneficial or potentially synergistic effect. The effects of the formula1 compounds on the IC50 (concentration of antimicrobial agent causing a50% reduction in pathogen replication or cytopathic effects in a givenassay), IC90 (concentration of antimicrobial agent causing a 90%reduction in pathogen replication or cytopathic effects in a givenassay), TC50 (concentration of antimicrobial agent causing a 50%reduction in cell growth or killing of 50% of cells in the absence ofthe pathogen in a given assay), TC90, LD50 (pathogen inoculum size thatcauses death of 50% of host subjects), LD90 therapeutic index (e.g., theTC50 divided by the IC50) and/or the MIC of the antimicrobial agent arethen determined, e.g., a decrease in the antimicrobial's IC50 or the MICby at least about 10%, at least about 20%, at least about 30%, at leastabout 40% or at least about 50%.

In an exemplary protocol, the capacity of a formula 1 compound topotentiate the effect of an antibacterial agent or antibiotic isconducted using a whole cell growth inhibition assay. The assay is runin the presence of both the formula 1 compound and the antibacterialagent, with the antibacterial agent at a concentration of about ⅕ theMIC of the antibiotic alone. When testing for a combination that can beused against methicillin resistant Staphylococcus aureus (“MRSA”) havinga MIC for methicillin of >100 μg/mL, the MRSA cells are grown in thepresence of 1-50 μg/mL of a formula 1 compound and 20 μg/mL ofmethicillin as follows.

A fresh inoculum of MRSA cells is grown at 35° C. in Mueller-HintonBroth (MHB) overnight and is then diluted about 1/50 in the same medium.After regrowth at 35° C. to an OD₆₀₀ of 0.2-0.4 (about 1-2 hrs.), thecells are diluted 1/1000 in MHB. In a microtiter plate, 50 μL of thisdiluted culture is combined with 50 μL MHB containing 10 μg/mL of theformula 1 compound and 40 μg/mL methicillin to give starting growthconditions of 5×10⁵ cfu/ml in MHB, with 5 μg/mL formula 1 compound and20 μg/mL methicillin. The plate is placed in a humidified 35° C.incubator for 5-24 hours, and cell growth or inhibition is read asturbidity (OD₆₀₀). Two positive and one negative controls for growth arecells grown in MHB alone, cells grown in MHB plus 20 μg/mL methicillinalone, and uninoculated MHB respectively. The capacity of the formula 1compound to detectably inhibit growth, e.g., inhibition of at leastabout 20%, at least about 30%, at least about 40%, at least about 50% orat least about 60% of the growth of the positive control withmethicillin, is determined in this assay. These results are compared togrowth of the MRSA in MHB alone and in MHB with 5 μg/mL formula 1compound to determine if the formula 1 compound alone appreciablyinhibits growth of MRSA.

Further evaluation of the effect of the formula 1 compound on the MIC ofan antibiotic such as methicillin is optionally evaluated in amicrodilution assessment of the MIC with methicillin for MRSA, e.g.,Lorian ed., Antibiotics in Laboratory Medicine, 3rd ed., pp. 72-75 1991.Also, a time-kill method, e.g., Eliopolous & Moellering, Ch. 13,Antibiotics in Laboratory Medicine, 3rd ed., Lorian ed., pp. 441-4441991, is optionally used to characterize whether the formula 1 compoundand antibiotic drug combination is bacteriostatic or bactericidal. Ascontrols, normal growth of the pathogen (without antibiotic or formula 1compound) and the effects of the antibiotic alone and the formula 1compound alone are assessed using the same inoculum culture as is usedin the combination time-kill test. Either bacteriostatic or bactericidaleffect can be determined. Additional assays for other antimicrobialagents and pathogens, e.g., antivirals, antifungals, antibacterials,viruses, fungi or bacteria, are optionally performed using antimicrobialcompounds disclosed herein or in cited references and using methodsdescribed herein and in cited references that are modified toincorporate the use of the formula 1 compounds and/or a variety ofsuitable antimicrobial agents, e.g., U.S. Pat. Nos. 6,306,880,6,303,797, 6,297,401, 6,180,679 and 5,883,074, Methods for AntimicrobialSusceptibility Testing/M11-A2, 1990, NCCLS, ISBN 1562380990 orPerformance Standards for Antimicrobial Susceptibility Testing: EighthInternational Supplement (1998), 1998, NCCLS, ISBN 156238337X, D. F.Smee et al., Antiviral Res. 2001 52(3):251-259, R. T. Sarisky et al., J.Clin. Virol. 2002 23(3):191-200, Y. C. Huang et al., Am. J. Perinatol.2001 18(3):141-146, J. Meletiadis et al., J. Clin. Microbiol. 200139(9):3402-3408, A. L. Barry et al., Eur. J. Clin. Microbiol. Infect.Dis. 1999 18(4):305-309. Such assays may comprise one or more of aradial diffusion assay, a plaque assay, a flow cytometry assay or ananimal infection assay, e.g., for animal survival or amelioration ofinfection, a tissue culture assay, e.g., for viral reverse transcriptaselevels, cytopathic effects or for levels of the pathogen or itsprotein(s), or another suitable assay described in the cited references.

Example 46

Domestic animals, e.g., cows, sheep, swine or goats, are dosed once ortwice with about 1 mg/kg, about 5 mg/kg, about 20 mg/kg or with about 50mg/kg of a formula 1 compound such as BrEA,3β,17β-dihydroxyandrost-5-ene or 3β,7β,17β-trihydroxyandrost-5-ene by aparenteral route, e.g., by subcutaneous or intramuscular injection, at1, 2, 3, 4, 5, 6, 7 or 14 days before the animals are to be transportedby, e.g., truck or train, from one location to another. The one or twodoses are administered on the same day or on different days within thetwo weeks before the animals are to be shipped and, for animals dosedtwo times, each dose is the same, e.g., two doses of doses 5 mg/kg ordoses 20 mg/kg, or different, e.g., the first dose is doses 20 mg/kg ordoses 50 mg/kg and the second dose is doses 1 mg/kg, doses 5 mg/kg ordoses 20 mg/kg. Each dose is administered at 1, 2, 3 or more sites. Theincidence and severity of stress, shipping fever, weight loss andinfection associated with transport is monitored. The capacity of theformula 1 compound to detectably reduce the incidence, severity or rateof progression of an unwanted condition is monitored and is optionallycompared to the effects of known agents for the same or similar uses.

In a related example, domestic animals are dosed with 5 mg/kg, 20 mg/kg,50 mg/kg or 100 mg/kg of a formula 1 compound orally, e.g., in feed, orparenterally at intervals of about 4 weeks, about 8 weeks, about 12weeks or about 16 weeks. Dosing optionally occurs over selected timeperiods, e.g., during the animals' entire lifespan, in the first 3-12months of the animals' life or about 1-6 months before the animal is tobe transported or otherwise expected to experience significant stress.The formula 1 compound is also optionally administered prophylacticallyto prevent or detectably reduce the rate of spread, the severity or theincidence of an anticipated or potential infection, e.g., a viralinfection such as a foot-and-mouth disease virus infection, a bacterialinfection or a prion infection. During dosing periods with the formula 1compound, the amount of antibiotic that the animals receive in theirfeed is optionally decreased, e.g., by about 10%, about 20%, about 30%,about 40%, about 50% or more during and after, e.g., about 1-16 weeksafter, the period of dosing has ended.

In another related example, animals are vaccinated against pathogeninfection using a formula 1 compound and an antigen(s). The formula 1compound is administered at 2, 3 or 4 different dosages over a range ofdosages, e.g., between about 0.2 mg/kg and about 100 mg/kg. A suitablepathogen or portion of pathogen is used, e.g., fungal, bacterial orviral cells (live, killed or attenuated) or particles may be used.Alternatively, the antigen may comprise protein(s), glycoprotein(s) oran antigenic fragment(s) thereof, and optionally an adjuvant with theantigen. The adjuvant may comprise an immune stimulatory complex,lipopolysaccharide, Freund's complete adjuvant, Freund's incompleteadjuvant, aluminum hydroxide or Montanide ISA 206. A dosage of a formula1 compound is administered as described above or elsewhere herein to theanimal at about the same time or within about 1, 2 or 3 days before orafter the antigen(s) is administered. The formula 1 compound isadministered to the animal on 1, 2, 3 or more occasions, typically onceor twice within 48 hours of vaccination or once on the same day theanimals are vaccinated. The effect of the formula 1 compound on theanimal's cellular and/or humoral immune response is optionallymonitored. Viral antigens or viral expression vectors and immuneresponse monitoring methods for foot-and-mouth disease virus vaccinationare optionally used in this embodiment.

The response of the vaccinated animals to a subsequent challenge withinfectious foot-and-mouth disease virus is generally determined. Theanimals are vaccinated on a single occasion or on two or more occasions,with the latter vaccinations optionally tested for a boost in the immuneresponse after an initial vaccination. The animals' antibody, cytokineand/or immune cell response to vaccination is generally monitored atabout 1-21 days after vaccination. One or more parameters may bemonitored, e.g., the development of a specific cytotoxic T cellclone(s), modulation of macrophage activity, circulating levels of IgMor an IgG such as IgG1, IgG2 or IgG3 that binds to the vaccinationpathogen or antigen(s), or resistance to a subsequent infectiouschallenge with the pathogen. Results obtained with animals vaccinatedwith and without the formula 1 compound are compared. Suitable controlssuch as formulation lacking the formula 1 compound or adjuvant lackingthe antigen are optionally used.

A foot-and-mouth disease virus, viral antigen(s) or an immunogenicfragment(s) thereof is optionally used in the vaccination. See, e.g., J.Bayry et al., Microbiol. Immunol. 1999 43:765-771, J. Chinsangaram etal., J. Virol. 1999 73:9891-9898, G. A. Mayr, et al., Vaccine 200119:2152-2162, M. J. Grubman et al., Vaccine 1993 11:825-829, P. W. Masonet al., Virology 1997 227:96-102, M. Amadori et al., Arch. Virol. 1992122:293-306 and C. C. Brown et al., J. Virol. 1996 70:5638-5641, A.Rodriguez et al., J. Gen. Virol. 1996 77(Pt. 9):2089-2096. Antigens andother pathogens that may be used in an animal vacination embodiment witha formula 1 compound include Aleutian mink disease virus, sheep borderdisease virus, equine borna disease virus, canine distemper virus,rabies virus, pseudorabies virus, Shope papilloma virus, canineparvovirus and hog cholera virus.

When a formulation that is suitable for parenteral delivery of theformula 1 compound to the animals is used, it optionally comprises alocal anaesthetic such as procaine or lidocaine, e.g., a formulationcomprising a formula 1 compound, 1, 2 or more excipients and about 0.01%w/w to about 2.5% w/w of the local anaesthetic.

Example 47

Human HIV-infected patients with impaired or negligible antigen specificimmune responses, cell mediated immune responses or delayed-typehypersensitivity immune responses are treated with16α-bromoepiandrosterone essentially as described herein. White bloodcells from the patients are obtained before the compound is administeredand at times after an initial treatment and a 2^(nd) treatment, e.g.,dosing of about 50-200 mg at day 1 and at day 19, with assays at about6-40 days after treatment, e.g., at day 8, 15, 22, 29 and 36. Thepatients' response to antigens such as HIV p24 and Candida antigen or tophytohemagglutinin is measured. Effects on the patients' antigenspecific immune responses or markers of cell mediated immunity ismeasured. The capacity of other formula 1 compounds to restore antigenspecific or cell mediated immune responses in immune suppressed subjectsis characterized in a similar manner.

Example 48

Antiglucocorticoid effects of formula 1 compounds. Effects of formula 1compounds and Hydrocortisone on Proliferation in the Absence of aMitogen. A series of tests is run in triplicate using BALB/c mousespleen cells to demonstrate the effect of the formula 1 compounds andhydrocortisone (“Hycort”) on cellular proliferation in the absence of amitogen. Cultures of spleen cells are prepared and steroids are addedat, e.g., 0.1, 0.5, 1, 5 μM. Suitable controls are used. Twenty fourhours after setup, about 50 μCi [³H]-thymidine is added to each cell.Four to six hours later, the cells are harvested and counted on ascintillation counter.

Spleen cells are obtained from normal BALB/c mice and prepared as asingle cell suspension at a concentration of about 1×10⁷ cells/ml inRPMI 1640 supplemented with 2 mM L-glutamine, 5×10⁻⁵ M2-mercaptoethanol, 20 μg/ml gentamycin-sulfate, and 1% Nutridona-NS(Boehringer-Mannheim). Individual aliquots of cells are then pulsed for30 minutes at 37° C. with selected concentrations of formula 1compounds. After pulsing, the cells are washed several times in balancedsalt solution, resuspended in fresh medium, and then dispensed into24-well culture plates with a stimulatory concentration of anti-CD3(e.g., Leo et al. Proc. Natl. Acad. Sci. U.S.A., 84:1374 (1987)). Aftera 24-hour incubation period, culture supernatants are harvested forassessment of proliferation or cytokine production, e.g., IL-2, IL-3 orIL-4 using, e.g., the method of Mossman (J. Immunol. Meth. 65:55(1983)). 100% control titers of IL-3, IL-2 or IL-4 from normalstimulated splenocytes are obtained, exemplary values may be about 640units/mL or IL-2 and 160 units/mL for IL-4.

Effects of formula 1 compounds and Hydrocortisone on Proliferation inthe Presence of a Mitogen. A series of spleen cell cultures is run usinga formula 1 compound and/or hydrocortisone with cell cultures to whichconcanavalin A is added. Preliminary tests on cultures to whichconcanavalin A is added at concentrations of 10.0, 5.0, 2.5 and 1.0ng/mL. All tests on the effects of invention compounds on culturesstimulated with concanavalin A are performed with concanavalin A at,e.g., about 2.5 ng/mL. A mitogen such as ConA generally increases cellproliferation and the GCS can decrease proliferation. Detectable partialor complete reversal of the inhibitor effects of hydrocortisone indicatean anti-glucorticoid effect by the formula 1 compounds.

Effect of formula 1 compounds on IL-3 production. Exemplary formula 1compounds are tested to determine whether their effect on the level ofthe cytokine IL-3 expression by spleen cells in tissue culture and fortheir capacity to reverse the effects of a GCS in IL-3 expression. Thespleen cell cultures are prepared in accordance with the general methodabove. After 30 hours the level of IL-3 in the supernatants of thecultures was measured using the IL-3 ELISA kit manufactured by EndoGenInc., Boston, Mass. A GCS such as hydrocortisone will generally suppressthe production of IL-3 and the invention compounds are examined fortheir capacity to modify this effect. The IL-3 expressed by cells inculture may be recovered from the media containing IL-3 by known methodssuch as single or sequential reverse-phase HPLC steps on a preparativeHPLC column. (See Urdal, et al., J. Chromatog. 296:171 (1984) and U.S.Pat. No. 5,128,450).

Example 49

Activity of formula 1 compounds in the treatment of symptoms orprogression of a skin condition. Conditions such as psoriasis areexamined using formula 1 compounds in a suitable animal model or inhuman patients. The compounds are administered to the subject by topicaladministration or by systemic administration, e.g., oral, buccal orsubcutaneous injection. In one animal model of psoriasis, varyingnumbers of naïve and memory CD4⁺ T cells from minor histocompatibilitymismatched mice are used to reconstitute scid/scid mice. Skin lesionswith characteristics of human psoriasis arises when no memory T cellsare coinjected, and coinjection of memory T cells diminishes thesymptoms (M. P. Schon et al., Nature Medicine 1999 3:183-188). Varyingamounts of formula 1 compounds, e.g., 0 to 100 mg/kg/day, are used inthe absence of memory T cells and with various numbers of memory T cellsto characterize their effects on both types of T cells to cause diseaseor to reduce it.

Groups of the mice (e.g., about 4-15 animals per group) are injectedsubcutaneously with one or more dosage of about 1, 2.5, 5, 10 or 20mg/kg of a formula 1 compound such as 16α-fluoroandrost-5-ene-17-one,7β-hydroxy-16α-fluoroandrost-5-ene-17-one,7α-hydroxy-16α-fluoroandrost-5-ene-17-one,17α-hydroxy-16α-fluoroandrost-5-ene or17β-hydroxy-16α-fluoroandrost-5-ene. The compound is administered oncedaily or once every 2, 3 or 4 days for 1, 2, 3, 4, 5 or 6 weeksbeginning either before or at about the time when untreated controlanimals have developed symptoms or skin lesions characteristic of thedisease. The formula 1 compounds can be used with other animal modelsand markers of psoriasis would be measured essentially as described.See, e.g., B. J. Nickoloff, J. Invest. Dermatol. Symp. Proc. 20005:67-73, M. P. Schon J. Invest. Dermatol. 1999 112:405-410, B. J.Nickoloff et al., Arch. Dermatol. 1999 135:546-552 and M. P. Schon etal., Nature Medicine 1997 3:183-188.

In a clinical dose ranging protocol, human patients suffering frompsoriasis are treated topically or systemically with a formula 1compound. Topical formulations would comprise about 1-20% w/w of aformula 1 compound in a suitable cream, gel or other topicalformulation, e.g., as disclosed herein. Treatment is initiated at theonset of symptoms or is administered to patients with preexistingpsoriatic lesions. The compound is administered twice per day, once perday or it is administered once or twice per day every other day, everythird day, every fourth day or every seventh day for about 2-24 weeks,e.g., about 4, 8 or 12 weeks. The clinical response to treatment ismonitored on one, two or more occasions during dosing and optionallyagain once or twice at 1 day to 6 weeks after dosing is completed.Treatment is optionally combined with another treatment(s), e.g.,corticosteroid, calcipotriol or salicylic acid. Patient response ismeasured using standard assessment methods, e.g., the Psoriasis ScalpSeverity Index for psoriasis of the scalp, the Psoriasis DisabilityIndex or by assessment of erythema, scaling, induration or size ofpsoriatic lesions at various times before, during and/or aftertreatment.

The response of animals or humans is optionally examined byhistopathological observation or other analysis, e.g., detectablyreduced inflammation or epidermal thickening, erythema, induration orfor a detectable modulation or reduction of inflammatory orpathology-associated cells, cytokines or markers, e.g., CD94⁺ T cells,CD69⁺T cells, CD45RO⁺ T cells, CD25⁺ T cells, IgE, IL-1α, IL-6, IL-8,IL-13, keratinocyte growth factor or transforming growth factor-α inskin or skin lesions from treated animals as compared to suitablecontrol animals. Modulation, e.g., reduction, of one or more markers orcell types associated with the severity or progression of disease ismonitored.

Example 50

Treatment of neurodegenerative conditions. Experimental allergicencephalomyelitis (EAE), is demyelinating disease of the central nervoussystem with many pathological and clinical similarities with multiplesclerosis (MS). EAE is thus a recognized animal model for humanautoimmune conditions such as MS. Formula 1 compounds16α-fluoroandrost-5-ene-17-one, 3β,7β,17β,-trihydroxyandrost-5-ene and16α-bromoepiandrosterone were tested for their capacity to delay theonset of EAE or to reduce its symptoms. Female SJL mice (5 animals pergroup) were immunized with 150 to 200 μg of PLP-139-151 peptide/CFA toinduce EAE. Starting 7 days before injection of the peptide, the animalswere given daily injections (s.c.) of the compounds (3.0 mg) in 0.1 mLvehicle, or vehicle alone for 33 days. The vehicle consisted of asuspension of the formula 1 compound in saline andcarboxymethylcellulose. 16α-Fluoroandrost-5-ene-17-one and3β,7β,17β,-trihydroxyandrost-5-ene significantly delayed the onset,reduced peak clinical score (from 5.2±0.6 to 2.8±1.8) and cumulativedisease index (>60%) of EAE, and prevented or significantly attenuatedrelapses. A lower dose (0.3 mg/mouse) or other routes (p.o.) ofadministration had less effect. The effect of 16α-bromoepiandrosteronewas not significant under these assay conditions. T cells from micetreated with 16α-fluoroandrost-5-ene-17-one and3β,7β,17β,-trihydroxyandrost-5-ene had significantly reduced PLP-139-151specific T cell proliferation responses and reduced numbers of TNF-αproducing cells in the CNS. These compounds limited the production ofautoimmune Th-1 associated cytokines, which is consistent withrestoration of a more normal Th-1/Th-2 immune balance and/or withreduction of inflammation in this model.

The efficacy of the formula 1 compounds to treat other autoimmuneconditions can be determined by incorporating their use with suitableanimal models or assay methods, e.g., collagen-induced arthritis as amodel for human autoimmune polyarthritis (e.g., L. K. Myers et al.,Clin. Immunol. 2002, 102:185-191, A. Matejuk et al., Endocrinology 2002,143:313-319, S. Thornton et al., J. Immunol. 165:1557-1563). The effectof the compounds on markers of inflammation such as TNFα, MIP-1β, IL-13,IL-15, IL-17 or IL-18, e.g., reduced expression or activity, isoptionally observed in any autoimmune or inflammatory condition.

Example 51

Human clinical use of the formula 1 compounds to treat delayed effectsof radiation exposure is exemplified as follows. Patients scheduled toreceive a radiation therapy for a malignancy or a related condition,e.g., breast cancer, prostate cancer or benign hyperstatic hyperplasia,begin the planned therapy. For patients scheduled to receive a totalradiation dose of about 2-200 Gy (e.g., about 130-150 Gy, about 144 Gyor about 180 Gy) in one dose or in two or more divided subdoses, at 24hours after initiation of the radiation therapy, about 1-8 mg/kg/day(about 50 mg, 100 mg, 200 mg or 400 mg per day) of a formula 1 compoundis administered to the patient using a daily or an intermittent dosingprotocol. The compound is administered by an intramuscular,subcutaneous, buccal or sublingual route. The intermittent dosingprotocol provides dosing the formula 1 compound once per day for 5consecutive days, followed by no dosing for 3 weeks and then againdosing the formula 1 compound once per day for 5 consecutive days,followed by no dosing for another 3 weeks. This treatment regimen ismaintained until 4-6 months after the planned radiation therapy hasended.

In related treatment protocols, the patient is dosed with about 1-8mg/kg/day of the formula 1 compound once every other per day over 5days, until 3 doses are administered to the patient, followed by nodosing for 6 weeks and then again dosing the formula 1 compound onceevery other per day over 5 days, until 3 doses are administered to thepatient, followed by no dosing for another 6 weeks. This treatmentregimen is maintained until 6-24 months after the planned radiationtherapy has ended.

In other related protocols, a dosing protocol as described in the twoparagraphs above is used, except that initiation of dosing is delayeduntil 1 week or until 1 or 2 months after completion of the plannedradiation protocol and the treatment regimen is maintained until 6-48months after the planned radiation therapy has ended.

In any of these treatment protocols, the patient is optionally monitoredfor the time of appearance of a radiation late effect, its severity. Thepatient is also optionally treated with other therapeutic agents, e.g.,analgesics (e.g., aspirin, ibuprofin, codeine or morphine),corticosteroids (e.g., prednisone or dexamethasome), antibiotics,antifungal agents, growth factors (e.g., erythropoietin, thrombopoietin,γ-interferon or IL-2), blood transfusion or surgery, as the clinicalsituation dictates.

Example 52

A buccal formulation containing 16α-fluoroandrost-5-ene-17-one for humanor veterinary applications was prepared as follows. Micronized16α-fluoroandrost-5-ene-17-one, PEG 3350, Cab-O—Sil™, Polyplasdone XL10™, Pearlitol™, and sodium lauryl sulfate were dispensed into a doublecone blender (Gemco) and blended for approximately 15 minutes. Three0.15 gram samples were collected from top, middle and bottom regions ofthe blend and assayed by HPLC for uniform drug content. Results from theHPLC assay for uniform drug content were obtained prior to continuingthe manufacture process. Blending was continued, if needed, until theblend contained 19% to 21% of 16α-fluoroandrost-5-ene-17-one by weightin selected samples. Magnesium stearate, sieved through a #40 screen,was then added to the mixture and blended for 5 minutes.

The uniform blend or mixture was then transferred to a doublepolyethylene bag and loaded into a tablet press hopper. Ten tablets(pillow shaped) were sampled at 15-minute intervals during thetabletting process to monitor thickness, weight and hardness of eachtablet. Samples of 35 tablets were taken at the beginning, middle, andend of the tablet compression run for testing. The tablets werecollected from the press in polyethylene bags and visually inspectedprior to packaging in 100 cc high density polyethylene (HDPE) roundbottles (38-400 finish) at 500 tablets per bottle. The tablets werestored at controlled room temperature (20°-25° C.).

Excipients used in the formulation were mannitol, (Pearlitol™, 200 μmdiameter granules, Roquette), which provided a matrix for separation ofdrug particles in the tablet and a compression aid to promote freeflowing of the drug blend into the tablet die. Crospovidone(Polyplasdone XL 10™, ISP Pharmaceutical), NF, was used as a dispersingagent and to facilitate tablet disintegration. PEG 3350 (SpectrumQuality Products, Gardena, Calif.), NF, was used as a wetting anddispersion agent. Sodium lauryl sulfate, NF, was used as a dispersionagent. Magnesium stearate (Spectrum Quality Products, Gardena, CA), NF,was used as a lubricant to facilitate ejection of tablets from the die.Amorphous silica dioxide (Cab-O-Sil, >98%, Cabot Corp.) was used as aglidant (flow enhancer) to promote free flowing of the drug blend intothe tablet die. Average tablet weight was 125 mg, with 90% of thetablets varying by less than 15% in weight and no tablets varying bymore than 25% in weight. The final composition of the tablets is shownbelow.

Component % w/w mg/tablet Total weight (g) 16α-fluoroandrost-5-ene- 1620 700 17-one Mannitol 72 90 3150 Crospovidone 7 8.75 306.2 Magnesiumstearate 2 2.5 87.5 PEG 3350 1 1.25 43.8 Sodium lauryl sulfate 1 1.2543.8 Cab-O-Sil ™ 1 1.25 43.8 Total 100% 125 mg 4375.1

Example 53

Synthesis of compounds is exemplified in the following examples. Meltingpoints were determined in open capillaries in an electrically heated andstirred Thiel-type bath and are uncorrected. Solvents were removed on arotary evaporator under water pump vacuum at 30° C. or less. Nuclearmagnetic resonance (NMR) spectra were taken on Bruker WP-200SY andAM-300 MHz spectrometers. Spectra were measured in deuterated chloroform(CDCl₃) using tetramethylsilane (60) as reference for ¹H NMR spectra andthe CDCl₃ triplet (δ77.0) for ¹³C spectra. Abbreviations are s(singlet), d (doublet), t (triplet), q (quartet), m (multiplet). AKratos MS-80RFA mass spectrometer was employed to determine the highresolution mass spectra (HRMS) of the compounds, and a Hewlett-PackardLC-MS, 1100 series was employed to determine the liquidchromatograph/mass spectra (LCMS) of the compounds. Spectral data wereincluded for some known compounds where such data were not previouslyreported. Reagents and solvents used were purchased from AldrichChemical Co., Milwaukee, Wis. For column chromatography, Aldrich brandsilica gel of 70-230 and 200-400 mesh size was used.

The glucuronides of 7-oxygenated androgens were synthesized by reactingthe appropriately protected 7-oxygenated derivative with bromoglucuronates followed by selective hydrolysis to remove acetateprotection on the glucose hydroxyls. Good yields were obtained whenacetylated glucuronides of steroids were prepared first, acetates werehydrolyzed selectively and the resulting Δ-5 steroid glucuronates wereoxygenated at the allylic 7-position.

Synthesis of β-pyranoside forms of steroid glucuronides (as theirtri-O-acetyl methyl esters) was carried out by condensation of aspecific, open hydroxyl group of the steroid, with 2, 3,4,-tri-O-acetyl-1-bromo-1-deoxy-αD-glucopyranuronate utilizing the wellknown Loenigs-Knorr reaction. Acid acceptors such as silver carbonate orcadmium carbonate in anhydrous benzene were used to promote the reactionand freshly pulverized dry calcium sulfate was included as internaldesiccant. Reactions were run in the dark (when silver carbonate wasused), at room temperature for 23 to 48 hours, during which time ˜40-60%conversion took place (identified by the ¹H-NMR spectrum of the reactionmixture). The acetylated glucuronide methyl esters were isolated eitherby crystallization or by column chromatography on silica gel. Fractionalcrystallization was the major technique employed for isolating theKoenigs-Knorr product from the reaction mixture and it was found to bequite satisfactory in providing high purity glucuronides. In cases whereisomeric ortho ester or other side products were formed in largerquantities, the desired products were purified by column chromatographyon silica gel.

The structures of acetylated glucuronide methyl esters were confirmed by¹H-NMR, ¹³C-NMR, and mass spectrometry. The anomeric proton signal (1H)of the glucose moiety appeared as a doublet at 4.58-4.69 ppm (J=8-8.4Hz) indicating 13-glucuronoside linkage. The position of the olefinic6-H of the 7-keto steroids, usually occurred at 5.65-5.70 ppm and showeda down field shift of 0.35-0.4 ppm from 6-H of non 7-keto steroids; thiswas another significant point in the characterization of 7-ketoglucuronides. β-Anomers of these steroid glucuronopyranosides weregenerally obtained in good to excellent yield when corrected forrecovered unreacted steroids.

Deacetylation of acetylated glucuronide methyl esters, with or without a7-keto group was performed with freshly prepared sodium methoxide inanhydrous methanol at ambient temperature yielding methyl esters ofsteroid glucuronides as the major product in generally good to excellentyield. ¹H-NMR spectra of deacetylated steroid glucuronides exhibited theanomeric proton as a doublet at 4.4-4.48 ppm (J=7.4-8.3 Hz) therebyconfirming β-linkage. High resolution mass spectra (EI-MS) of steroidglucuronides helped confirm the structure of the products, but theexpected molecular ion was absent. The molecular ion was obtained as asodium adduct base peak in the FAB-MS spectra of the steroidglucuronides. Finally, allylic oxidation at the 7 position of Δ5-steroidglucuronides 8 and 12 was performed utilizing the aerobic oxidationprocedure of described in Marwah and Lardy, U.S. Pat. No. 5,869,709(1999), herein incorporated by reference in its entirety, in thepresence of N-hydroxy phthalimide and a radical initiator to afford thecorresponding 7-oxo derivatives of steroid glucuronides 9 and 13.

Molecules with small and long chain alkyl ethers were also synthesized.The methyl ether (20) of 7-oxo DHEA (2) was prepared in 67% yield byallylic oxidation of 3β-methoxy-DHEA (19) which was obtained by heating3β-tosyl-DHEA to reflux in anhydrous methanol in 94% yield. Furtherreduction of the product 20 with sodium borohydride inmethanol-dichloromethane (usually 90:10) at 0-5° C. in presence ofcerium (III) chloride heptahydrate afforded3β-methoxyandrost-5-en-7β,17β-diol (24). The 17-keto group of 3β-methoxyDHEA (19) was protected as ethylene ketal, and subsequent oxidation ofthe ketal derivative afforded the 7-oxo compound (21). The 7-oxo groupof product 21 was further subjected to reduction with sodium borohydridein methanol at room temperature and a mixture of isomeric 7α- and7β-hydroxy derivatives (22, 23) were formed, and were separated easilyby column chromatography. Reduction of 3β-Methoxy DHEA (19) at position17, at 0-5° C. afforded 3β-methoxy-17β-hydroxyandrost-5-ene (27) in 86%yield, which in turn was oxidized at the 7 position using N-hydroxyphthalimide, oxygen and a radical initiator in refluxing acetone toafford product 28 (53% yield).

3β-t-Butyl ether of 7-oxoDHEA (31) was prepared in 87% yield byoxidizing 3β-t-butyl-DHEA (30) using pyridinium dichromate andN-hydroxyphthalimide, a new, simple and high yielding procedure.3β-t-ButylDHEA was synthesized in 67% yield by the procedure ofArmstrong et al. (Armstrong et al., Tet. Lett., 29:2483-2486 (1988),incorporated herein by reference in its entirety,) using DHEA andt-butyl trichloroacetimidate in a mixture of dichloromethane andcyclohexane.

According to the methods of the present invention, a mixture ofdiastereomeric 7-methoxy ethers (26) was prepared in a simple, shortprocedure, which unexpectedly resulted in very good yields (73%) bystirring 7α-bromo-DHEA (25) in methanol and silica gel at roomtemperature for 2 hours. No attempt was made to synthesize thesederivatives in pure α and β forms.

A long, straight chain alkyl ether (39) was prepared in 83% yield from3β-dodecanoxyDHEA by allylic oxidation of 3β-dodecanosyl-DHEA (38) usingpyridinium dichromate and N-hydroxyphthalimide as mentioned above.3β-Dodecanoxy DHEA (38) was synthesized using a standard procedure ofrefluxing 1-bromododecane and DHEA solution in tetrahydrofuran inpresence of sodium hydride.

Etherification of DHEA utilizing ethyl vinyl ether in dichloromethaneand in presence of p-toluene sulfonic acid, at room temperature for 6hours afforded 3β-ethoxy ethyl ether of DHEA (40) in 84% yield based on55% conversion. The product 40 was subjected to aerobic oxidation inpresence of N-hydroxyphthalimide and a radical initiator in refluxingacetone to obtain 3β-(1′-ethoxy)ethoxyandrost-5-en-7,17-dione (41).

3β-tetrahydropyranyl ether of 7-oxo-DHEA (37) was made to test theeffect of a saturated pyran ring having two stereogenic centers thusgenerated, on the induction of thermogenic enzymes. This compound waseasily formed utilizing dihydropyran and pyridinium p-toluene sulfonatein anhydrous dichloromethane and was stable to most non-acidic reagents.

Silyl protected ethers especially t-butyldimethylsilyl (TBDMS) ether,(32, 33, 35 and 36) substituted at hydroxyls 3, 3 and 17, 7, and 7 and17 positions of the steroid molecule were also synthesized. TBDMS ethersare quite stable to a variety of organic reactions and have goodstability toward base. Their synthesis involved use of t-butyldimethylsilyl chloride and imidazole in dimethyl formamide.Dichloromethane was not appropriate for the synthesis of these specific7-substituted derivatives because its acidic character caused theformation of dienes.

In another embodiment of the present invention, carbonate substitutionon hydroxyls at 3 and 17 position of the corresponding sterols with7-position oxygenated (keto or hydroxyl) in the steroid molecule wasperformed and its effect investigated. Various alkyl carbonates such asmethyl (42), ethyl (44), allyl (43), isobutyl (45), and octyl (47) aswell as tricyclic fluorenyl carbonates of the enzyme activator were madeby standard procedures which involved a slow addition of respectivechloroformates to the ice cold solution of steroid substrates inpyridine. Conversion was total in most of the cases and product yieldswere generally high. Carbonates 42 and 43 were isolated by columnchromatography on silica gel. Some of the carbonate derivatives of thesteroids (42, 44, 47) were subjected to borohydride reduction inmethanol-dichloromethane with cerium (III) chloride heptahydrate at 0-5°C. and 7β- and 17β-hydroxy carbonate derivatives (49-51) were isolatedin good yields.

The determination of the structure of the 7-oxygenated alkyl ethers aswell as carbonates and sterochemical assignments were made on the basisof their ¹H NMR and ¹³C NMR spectroscopic data, high resolution massspectroscopy and LC-MS studies. Reaction products, as well as someprecursors, when known, were also characterized by comparison ofspectroscopic data with those available from the literature. Proton NMRof 6-olefinic hydrogen of all 7-oxo derivatives showed a characteristicabsorption at δ 5.7-5.75 with a corresponding ¹³C absorption at about200-202 ppm. On the other hand, 7-hydroxyl derivative showed differentchemical shift values for 6-olefinic protons. In case of 7β-hydroxycompound the olefinic 6α-proton appeared high field and around 5.3 ppm,whereas 7α-hydroxy compound showed a doublet for 6β-olefinic protondownfield and around 5.6 ppm.

Assays of compounds for their ability to induce liver mitochondrialglycerophosphate dehydrogenase and cytosolic malic enzyme when fed torats have been described. Lardy et al., Steroids, 63:158-165 (1998); Suand Lardy, J Biochem, 110:207-213 (1991), which are incorporated hereinby reference in their entirety. In addition to the control rats thatreceived no steroid supplement, each experiment included a group thatreceived DHEA or 7-oxo-DHEA-acetate (usually at 0.04%-0.06% of thediet). Because of variation in enzyme activity of both control andtreated groups from one experiment to another the activities arereported relative to the enzyme activities of the control group.Compounds are considered active in this assay if they enhance theactivity of these thermogenic enzymes to greater than about 150% of thatin livers of control rats.

Example 1

General Synthetic Procedure: Preparation of β-Pyranoside Forms ofSteroid Glucopyranosides (as tri-acetylated methyl esters, 7, 10, 11,14, 15, 17) Steroid (10.0 mmol),methyl-2,3,4-tri-O-acetyl-1-bromo-1-deoxy-α-D-glucopyranuronate (10.0mmol) and freshly pulverized dry calcium sulfate (2.0 g) were taken upin dry benzene (150 mL). Dry silver carbonate (freshly prepared, 20.0mmol) was added to the reaction mixture which was subsequently stirredat room temperature in the dark. Additional quantities of glucuronate (5mmol) and silver carbonate (10 mmol) were added after 12 and 24 h. Thereaction mixture was stirred for 48 to 72 h depending upon the nature ofthe steroid, and then worked up. The reaction mixture was filtered on abed of celite and the clear filtrate was evaporated to dryness. Theproduct was isolated from the crude solid either by crystallization orby chromatography on alumina or silica gel. These synthetic procedureshave been described in P. Marwah et al., Steroids 2001 66:581-595.

Example 2 Methyl2,3,4-tri-O-acetyl-1-O-(17-oxoandrost-5-ene-3β-yl)-β-D-glucopyranosiduronate(7)

A mixture of DHEA (1) andmethyl-2,3,4-tri-O-acetyl-1-bromo-1-deoxy-α,D-glucopyranosiduronate, drycalcium sulfate and silver carbonate in benzene was stirred for 48 h atroom temperature. The NMR of the reaction mixture showed 60% conversionof the starting material to the product. After the usual work-up andsubsequent crystallization of the crude product, twice, fromacetone-hexane, product 7 was obtained as a pure white solid (61%, basedon 60% conversion), m.p. 191-92° C. ¹H NMR (CDCl₃ 200 MHz): δ 5.39 (1H,d, J=5.2 Hz, 6-H), 5.24 (2H, m, 3,4-H (glu)), 4.98 (1H, dd, J=8.0, 10.0Hz, 2-H (glu)), 4.66 (1H, d, J=8.0 Hz, 1-H (glu)), 4.02 (1H, d, J=10 Hz,5-H (glu)), 3.75 (3H, s, OCH₃), 3.52 (1H, m, 3α-H), 2.10, 2.05 (9H, 2s,OCOCH₃), 1.02 (3H, s, 19-CH₃), 0.89 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 220.7 (C-17), 170.0, 169.2, 169.0, 167.5(C═O, acetate, ester), 140.3 (C-5), 121.1 (C-6), 99.2 (C-1 (glu)), 79.6(C-3), 72.9, 72.0, 70.15, 69.3 (C-2-5 (glu)), 52.8, 51.5, 50.0, 47.4,38.4, 37.2, 37.0, 35.6, 31.6, 31.1, 29.6, 22.2 (C-1,2,4,7-16 and OCH₃),21.0, 20.9, 20.8, 19.5, 14.4 (3×OCOCH₃, C-18, C-19).

Example 3

Methyl2,3,4-tri-O-acetyl-1-O-(7,17-dioxoandrost-5-ene-3β-yl)-β-D-glucopyranosiduronate(10). The product 10 was prepared by the glucuronidation of3β-hydroxyandrost-5-ene-7,17-dione (2) and the reaction mixture wasstirred at room temperature in the dark for 72 h. The crude product waspurified by chromatography on alumina and eluted with ethylacetate-hexane (15:85, v/v). The first fraction eluted from the columnwas crystallized from methanol and identified asandrosta-3,5-diene-7-17-dione (15%, based on 69% conversion), m.p.167-68° C. ¹H NMR (ODCl₃, 200 MHz): δ 6.19 (2H, m, 3,4-H), 5.65 (1H, s,6-H), 1.15 (3H, s, 19-CH₃), 0.90 (3H, s, 18-CH₃). Further elution from a30:70 mixture of ethyl acetate-hexane afforded a thick syrupy materialwhich was triturated with diethyl ether and cooled in the refrigeratorfor 2 h, to afford white crystalline compound 10, m.p. 206-8° C. (25%,based on 69% conversion).

¹H NMR (CDCl₃, 200 MHz): δ 5.75 (1H, s, 6-H), 5.24 (2H, m, 3,4-H (glu)),4.98 (1H, t, J=8.3 Hz, 2-H (glu)), 4.65 (1H, d, J=8.3 Hz, 1-H (glu)),4.03 (1H, d, J=9.9 Hz, 5-H (glu)), 3.75 (3H, s, OCH₃), 3.65 (1H, m,3α-H), 2.04, 2.0 (9H, 2s, OCOCH₃), 1.20 (3H, s, 19-CH₃), 0.91 (3H, s,18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 220.4 (C-17), 201.1 (C-7), 170, 169.4,169.2, 167.1 (C-acetate, ester), 165.1 (C-5), 126.2 (C-6), 100.0 (C-1(glu)), 78.4 (C-3), 72.9, 72.1, 71.4, 69.4 (C-2-5 (glu)), 21.0, 17.6,14.1 (3×OCOCH₃), 53.2, 48.2, 45.8, 44.6, 39.6, 39.0, 39.2, 36.2, 35.6,31.0, 29.0, 24.3 (C-1, 2, 4, 8-16, OCH₃).

FAB m/z: 641.1 (M+23, 100%), 499 (M+1, −60, −60, 1%), 285 (M+1, −334,4%). A final fraction eluted from the same composition of the eluentafforded the starting 3β-hydroxyandrost-5-ene-7,17-dione (2, 31%).

Example 4 Methyl2,3,4-tri-O-acetyl-1-O-(3β-acetoxyandrost-5-ene-17β-yl)-β-D-glucopyranosiduronate(11). After 48 hours of stirring, 3β-acetoxyandrost-5-ene-17β-ol (3)with acetobromoglucuronate methyl ester as described above, the ¹H NMRspectrum of the reaction mixture showed the presence of equal amounts of3 and the product steroid glucopyranoside 11. Prolonged stirring beyond48 h did not alter the product ratio. The reaction mixture was processedand the resultant thick mass was heated to dissolve in a mixture ofacetone-hexane. The solution was allowed to stand at room temperaturefor 4 h and the white crystalline compound 11 was collected and dried.¹H NMR spectrum of the crystalline solid confirmed the purity andstructure of product 11, yield 94.8% (based on 50% conversion ofstarting steroid), m.p. 187-9° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.36 (1H, d, J=5.2 Hz, 6-H), 5.23 (2H, m,3,4-H (glu)), 5.02 (1H, t, J=8.4 Hz, 2-H (glu)), 4.60 (1H, d, J=8.4 Hz,1-H (glu)), 4.60 (1H, m, 3α-H), 3.99 (1H, d, J=10 Hz, 5-H (glu)), 3.75(3H, s, OCH₃), 3.59 (1H, t, J=8.7 Hz, 17α-H), 2.07, 2.05, 2.04, 2.03(12H, 4s, OCOCH₃), 1.03 (3H, s, 19-CH₃), 0.74 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 170.53, 170.23, 169.35, 169.07, 167.26 (C=0acetate and ester), 139, 67 (C-5), 122.24 (C-6), 101.43 (C-1 (glu)),90.03 (C-3), 73.82, 72.54, 72.15, 71.42, 69.52 (C-17 and C-2, 3, 4, 5(glu)), 52.83, 50.95, 50.05, 42.81, 38.07, 37.35, 37.0, 36.61, 31.6,31.37, 28.56, 27.72, 23.35 (C-1, 2, 4, 7-16, and OCH₃), 21.41, 20.72,20.63, 20.50, 19.32, 11.45 (acetate-CH₃, C-18, C-19).

Example 5

Methyl-2,3,4-tri-O-acetyl-1-O-(3β-acetoxyandrost-5-ene-7-oxo-17β-yl)-β-D-glucopyranosiduronate(14). Glucuronidation of 3β-acetoxy-17β-hydroxyandrost-5-ene-7-one (4)was performed similarly and the reaction mixture was worked up afterstirring for 48 h. ¹H NMR analysis of the filtrate showed 60% of thestarting material and 40% of the product. The crude mixture was treatedwith hexane and crystallization at room temperature afforded a whitesolid that was filtered and air dried. The crystalline white solid wasfound by ¹H NMR to be pure compound 14. M.P. 122-25° C., yield 78.5%(based on 40% conversion).

¹H NMR (CDCl₃, 200 MHz): δ 5.71 (1H, d, 6-H), 5.23 (2H, M, 3,4-H (glu)),5.02 (1H, t, J=8.0 Hz, 2-H (glu)), 4.72 (1H, m, 3α-H), 4.58 (1H, d,J=8.0 Hz, 1-H (glu)), 4.0 (1H, d, J=10.0 Hz, 5-H (glu)), 3.76 (3H, s,OCH₃), 3.6 (1H, t, J=8.0 Hz, 17α-H), 2.06, 2.046, 2.025, 2.02 (12H, 4s,OCOCH₃), 1.22 (3H, s, 19-CH₃), 0.74 (3H, s, 18-CH₃).

FAB m/z: 685.1 (M+23, 100%), 625.1 (M+23, −60, 6%), 269.2 (M, −334, −60,8%).

Example 6

Methyl-2,3,4-tri-O-acetyl-1-O-(3β,17β-diacetoxyandrost-5-ene-7,3-yl)-β-D-glucopyranosiduronate(15). The reaction mixture containing3β,17β-diacetoxyandrost-5-ene-7β-ol (5), acetobromoglucuronate methylester, freshly dried calcium sulfate and silver carbonate in anhydrousbenzene was stirred for 48 h at room temperature during which time 60%conversion of the starting steroid was detected on the basis of the NMRof the reaction mixture. The mixture was filtered over a small bed ofcelite and clear filtrate was evaporated to dryness under vacuum at 30°C. The resulting solid was chromatographed on a silica gel column usingethyl acetate-hexane (30:70, v/v) as an eluent to afford first thestarting steroid followed by the product 15. Crystallization of steroidglucopyranoside 15 from methanol afforded a white crystalline compoundm.p. 230-31° C., yield 54.5% (based on 61% conversion).

¹H NMR (CDCl₃, 200 MHz): δ 5.55 (1H, m, 6-H), 5.22 (2H, m, 3,4-H (glu)),5.02 (1H, dd, J=8.0 Hz, 9.8 Hz, 2-H (glu)), 4.69 (1H, d, J=8.0 Hz, 1-H(glu)), 4.6 (1H, t, J=8.3 Hz, 17α-H and 1H, m, merged, 3α-H), 3.99 (1H,d, J=9.8 Hz, 5-H (glu)), 3.9 (1H, dm, J=8.0 Hz, 7α-H), 3.76 (3H, s,OCH₃), 2.04, 2.02, 2.00 (15H, 3 s, OCOCH₃), 1.05 (3H, s, 19-CH₃) 0.76(3H, s, 18-CH₃).

FAB m/z: 729.1 (M+23, 100%), 669.1 (M+23, −60, 5%), 313.2 (M+1, −334,−60, 15%) 253.2 (M+1, −334, −60, −60, 7%).

Example 7

Methyl-2,3,4-tri-O-acetyl-1-O-(3β-acetoxy-17-oxoandrost-5-ene-7α-yl)-β-D-glucopyranosiduronate(17). The reaction mixture comprised of3β-acetoxy-7α-hydroxyandrost-5-ene-17-one (6), acetobromoglucuronatemethyl ester, freshly dried calcium sulfate, silver carbonate and acatalytic amount (0.5 mole %) of silver triflate in anhydrous benzenewas stirred for 48 h at room temperature in the dark. A total of 60%conversion of the starting steroid was detected on the basis of the NMRof the reaction mixture. The mixture was filtered over a small bed ofcelite and clear filtrate was evaporated to dryness. The product steroidglucopyranoside 17 was obtained in 34.8% yield (based on 60% conversion)by crystallization from acetone-hexane, m.p. 240-42° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.64 (1H, d, J=5.37 Hz, 6-H), 5.24 (2H, m,3.4-H (glu)), 4.96 (1H, t, J=7.81 Hz, 2-H (glu)), 4.68 (1H, d, J=7.81Hz, 1-H (glu)), 4.58 (1H, m, 3α-H), 4.02 (1H, d, J=9.77 Hz, 5-H (glu)),4.02 (1H, m, 7(3-H), 3.74 (3H, s, OCH₃), 2.047 (3H, s, OCOCH₃), 2.034(3H, s, OCOCH₃), 2.03 (3H, s, OCOCH₃), 2.02 (3H, s, OCOCH₃), 1.03 (3H,s, 19-CH₃) 0.84 (3H, s, 18-CH₃).

FAB m/z: 685.1 (M+23, 50%), 329 (M+1, −334, 16%), 269 (M+1, −60, −334,6.9%).

Alkaline hydrolysis of steroid acetylated glucuronide methyl esters.

Example 8 Methyl1-O-(17-oxoandrost-5-ene-3β-yl-β-D-glucopyranosiduronate (8)

Methyl2,3,4-tri-O-acetyl-1-O-(17-oxoandrost-5-ene-3β-yl)-β-D-glucopyranosid-uronate(7) (0.5 g, 0.83 mmol) was placed in a flame-dried flask with 50.0 mL offreshly dried and distilled methanol. A freshly prepared solution (0.65mL) of sodium methoxide (prepared by dissolving 0.14 g sodium in 5 mL ofmethanol) was added to the solution and the mixture was stirred at roomtemperature for 3 h. Solvent was evaporated and the residue was taken upin distilled water (25 mL). The excess of base was neutralized withsolid carbon dioxide or dilute acetic acid and the aqueous layer wassaturated with sodium chloride. A white precipitated solid, thus formed,was extracted with ethyl acetate and the organic layer washed withbrine, dried over magnesium sulfate and filtered. Solvent was removedand the residue, after trituration with ether, afforded a white solidwhich was filtered and dried. Recrystallization from acetone-hexaneyielded 0.23 g (75.8%), m.p. 208-10° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.41 (1H, d, J=4.6 Hz, 6-H), 4.45 (1H, d,J=8.3 Hz, 1-H (glu)), 3.92-3.24 (5H, m, 2-5H (glu) and 3-αH), 3.83 (3H,s, OCH₃, 1.04 (3H, s, 19-CH₃), 0.89 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 221.2 (C-17), 170.0 (ester C═O), 140.9(C-5), 121.8 (C-6), 101.4 (C-1 (glu)), 79.4 (C-3), 77.6, 74.3, 72.3,71.8 (C-2-5 (glu)), 53.1, 52.2, 50.7, 48.1, 39.2, 37.4, 37.0, 36.2,31.6, 31.2, 29.6, 22.2, 20.5 (C-1, 2, 4, 7-16 and OCH₃), 19.4, 14.6(C-18, 19).

Example 9

Methyl 1-O-(3β-acetoxyandrost-5-ene-17β-yl)-β-D-glucopyranosiduronate(12). To a solution of compound 11 (2.0 g. 3.08 mmol) in anhydrousmethanol (100 mL), 1.3 mL of a freshly prepared standard solution ofsodium methoxide (prepared as above), was added under argon. After 1 hstirring at room temperature, the methanol was distilled off at 25-30°C., the residue was taken up in cold water and neutralized with eithersolid carbon dioxide or dilute acetic acid. The aqueous layer wassaturated with sodium chloride and the solid was extracted withdichloromethane. The organic layer was filtered, washed with saturatedbicarbonate, dried over magnesium sulfate, and filtered. On triturationwith ether the product 12 was obtained as pure white crystals (1.4 g,87%), m.p. 192-5° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.37 (1H, d, J=5.5 Hz, 6-H), 4.6 (1H, m,3α-H), 4.41 (1H, d, J=8.2 Hz, 1-H (glu)), 3.83 (3H, s, OCH₃, 3.88-3.38(5H, m, 17α-H, 2, 3, 4, 5-H (glu)), 2.038 (3H, s, OCOCH₃), 1.03 (3H, s,C-19), 0.82 (3H, s, C-18).

¹³C NMR (DMSO, 300 MHz): δ 170.0, 169.8 (C═O, ester, acetate), 140.5(C-5), 122.3 (C-6), 104.3 (C-1 (glu)), 89.1 (C-3), 76.8, 76.2, 74.4,74.2, 71.1 (C-17, C-2, 3, 4, 5 (glu)), 21.0 19.2, 11.2 (C-18, C-19,acetate-CH₃).

FAB m/z: 559.2 (M+23, 100%), 499.2 (M+23, −60, 12%), 329 (M+1, −208,30%), 269 (M+1, −208, −60, 5%).

Example 10 Methyl1-O-(3β,17β-diacetoxyandrost-5-ene-7β-yl)-β-D-glucopyranosiduronate (16)

To a solution of compound 15 (0.19 g, 0.27 mmol) in dry methanol (10.0mL) was added 0.2 mL of a solution of sodium methoxide prepared asdescribed above. The solution was stirred at room temperature for 1 h.Methanol was removed at 25° C. and the resultant mass was taken up in 10mL of cold distilled water. Dilute acetic acid was added to neutralizethe excess base. The aqueous layer was extracted with ethyl acetate,washed with water and brine, and dried over magnesium sulfate. Ethylacetate was completely evaporated under vacuum at 25° C. and theresidue, on trituration with ether, afforded a white solid which wasfiltered, dried and identified as methyl1-O-(3β,17β-diacetoxyandrost-5-ene-7β-yl)-β-D-glucopyranosiduronate (16)(0.1 g, 64%), m.p. 140-42° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.59 (1H, s, 6-H), 4.68-4.50 (1H, t, J=8.5Hz, 17α-H and 1H, m, 3α-H), 4.48 (1H, d, J=7.4 Hz, 1-H (glu)), 4.00-3.3(5H, m, 2, 3, 4, 5-H (glu)+7α-H), 3.82 (3H, s, OC₃), 2.046 (6H, s,OCOCH₃), 1.06 (3H, s, 19-CH₃), 0.80 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 172.2, 171.2, 170.0 (C=0 acetate, ester),144.8 (C-5), 124.4 (C-6), 101.88 (C-1 (glu)), 82.94, 82.60 (C-3, 17),75.61, 74.14, 73.31, 71.23 (C-7, and 2, 3, 4, 5 (glu)), 52.68 (C-ester),50.64, 48.61, 37.9 (C-8, 9, 14), 42.93, 36.19 (C-10, 13), 37.83, 36.62,27.66, 26.0, 20.9 (C-1, 2, 4, 11, 12, 15, 16), 21.38, 21.19(CH₃-acetate) 18.83, 11.84 (C-18, 19).

HRMS m/z: 372.2274 for C₂₃H₃₂O₄ requires 372.2300 (M⁺, −208, 4%),330.2213 for C₂₁H₃₀O₃ requires 330.2195 (M⁺, −191, −59, 100%), 312.2132for C₂₁H₂₈O₂ requires 312.2089 (M⁺, −208, −60, 20%), 271 (M⁺, −191, −59,−59, 8%), 253.1972 for C₁₉H₂₅ requires 253.1956 (M⁺, −208, −60, −60,16%).

FAB m/z: 729.1 (M+23, 100%), 669.1 (M+23, −60, 5%), 609 (M+23, −60, −60,20%), 313 (M, −333, −60, 18%), 253 (M, −60, −60, −333, 8%).

Example 11

General Procedure for Allylic Oxidation of Steroid Glucuronides. Asolution of N-hydroxyphthalimide (5.0 mmol) and dibenzoyl peroxide (0.01g) in acetone-ethyl acetate (1:0.5, 75.0 mL) was brought to reflux, andthen a filtered hot solution of steroid methyl glucuronate (2.5 mmol) inacetone (20.0 mL) and dibenzoyl peroxide (0.01 g) were added. A slowstream of compressed air was passed into the solution and the mixturewas heated to reflux for 12-16 h (checked with TLC). Solvent was removedcompletely and the resultant mass was taken up in toluene and theprecipitated N-hydroxy phthalimide was filtered off. The organic layerwas washed thoroughly with saturated sodium bicarbonate solutionfollowed by water and brine and then dried over magnesium sulfate. Thesolvent was evaporated and the residue was chromatographed on silica gelusing acetone-hexane (1:1, v/v) and crystallized from acetone-hexane.

Example 12

Methyl 1-O-(7,17-dioxoandrost-5-ene-3β-yl)-β-D-glucopyranosiduronate(9). From compound 8, compound 9 was obtained, using the above generalprocedure, as white crystals, m.p. 203-5° C. (decomp.), yield 29%.

¹H NMR (CDCl₃, 200 MHz): δ 5.76 (1H, s, 6-H), 4.47 (1H, d, J=7.9 Hz, 1-H(glu)), 3.83 (3H, s, OCH₃), 3.95-3.35 (5H, m, 2-5H (glu) and 3α-H), 1.22(3H, s, 19-CH₃), 0.90 (3H, s, 18-(CH₃).

¹³C NMR (DMSO, 300 MHz): δ 219.12 (C-17) 200.48 (C-7), 169.54 (esterC=0), 166.3 (C-5), 125.23 (C-6), 101.3 (C-1 (glu)), 76.38, 75.79, 75,31, 73.0, 71.7 (C-2-5 (glu) and C-3), 16.93, 13.43 (C-18, 19), 51.83,49.30, 47.23, 44.98, 43.69, 38.64, 38.14, 35.80, 35.0, 29.2, 24.0, 20.02(C-1, 2, 4, 8-16, and OCH₃).

FAB m/z: 515.2 (M+23, 4%), 455.3.2 (M+23, −60, 9%), 413.2 (M, −60, −18,−1, 100%), 241.8 (M, −208, −28, −15, 2%).

Example 13

Methyl1-O-(3β-acetoxy-7-oxoandrost-5-ene-17β-yl)-β-D-glucopyranosiduronate(13). White solid 13 was prepared from 12, using the above generalprocedure; yield 38%, m.p. 118-20° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.71 (1H, s, 6-H), 4.72 (1H, m, 3α-H), 4.39(1H, d, J=8.2 Hz, 1-H (glu)), 3.84 (3H, s, OCH₃), 3.88-3.27 (5H, m,17α-H, and 2, 3, 4, 5-H (glu)), 2.06 (3H, s, OCOCH₃), 1.23 (3H, s,19-CH₃), 0.83 (3H, s, 18-CH₃).

¹³C NMR (DMSO, 300 MHz): δ 200.5 (C-7), 169.0, 169.2 (C═O acetate,ester): 165.0 (C-5), 125.8 (C-6), 104.1 (C-1 (glu)), 87.5 (C-3), 76.0,75.37, 73.6, 71.54 (C-17 and C-2, 3, 4, 5 (glu)), 21.0, 16.79, 11.30(C-18, 19, acetate CH₃), 51.8, 48.8, 45.0, 44.6, 43.3, 38.0, 37.5, 35.5,28.7, 27.0, 25.3, 20.0 (C-1, 2, 4, 8-16 and OCH₃).

Ether derivatives.

Example 14

3β-Methoxyandrost-5-ene-7,17-dione (20). A solution of3β-tosyloxyandrost-5-ene-17-one (18) (4.0 g. 9.05 mmol) in anhydrousmethanol (120 mL) was refluxed for 2 h. The reaction mixture wasconcentrated under vacuum. On cooling, a white solid3β-methoxyandrost-5-ene-17-one (19) was obtained. Recrystallization frommethanol afforded shining crystals (2.57 g. 94%), m.p. 133-5° C. (litm.p. 118-120° C. aq. MeOH).

¹H NMR (CDCl₃, 200 MHz): δ 5.39 (1H, d, J=5.3 Hz, 6-H), 3.37 (3H, s,OCH₃), 3.09 (1 h, M. 3α-H), 1.04 (3H, s, 19-CH₃), 0.90 (3H, s, 18-CH₃).

Air was passed through a refluxing solution of 19 (2.0 g; 6.62 mmol) andN-hydroxyphthalimide (1.5 g, 9.2 mmol) in a mixture of acetone-ethylacetate (1:1, 50 mL). 1,1′-azobis (cyclohexanecarbonitrile) (0.08 g) wasadded to the reaction mixture (Foricher et al.,

U.S. Pat. No. 5,030,739 (1991), incorporated herein by reference in itsentirety) and the reaction continued for 10-12 h. After the usual workup as described in the general oxidation procedure, the crude productwas dissolved in pyridine (5.0 mL) and stirred with acetic anhydride(3.0 mL) at room temperature for 3-4 h. The reaction mixture was pouredinto water and stirred for 2-3 h. The steroid was extracted with tolueneand the organic layer washed with saturated bicarbonate solution andwater. Toluene was distilled off and the residue was chromatographed onsilica gel (ethyl acetate-hexane, 25:75, v/v) and crystallized frommethanol. 3β-methoxyandrost-5-ene-7,17-dione (20) was obtained in 67%yield (1.4 g), m.p. 227-29° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.76 (1H, d, J=1.8 Hz, 6-H), 3.39 (3H, s,OCH₃), 3.2 (1H, m, 3a−H), 1.22 (3H, s, 19-CH₃), 0.90 (3H, s, 18-CH₃.

¹³C NMR (CDCl₃, 300 MHz): δ 220.27 (C-17), 200.99 (C-7), 166.22 (C-5),125.85 (C-6), 78.79, 55.91, 50.05, 45.69, 44.26 (C-3, 8, 9, 14, OCH₃),47.80, 38.72 (C-10, 13), 38.65, 36.13, 35.58, 30.67, 27.51, 24.12, 20.52(C-1, 2, 4, 11, 12, 15, 16), 17.36, 13.71 (C-18, 19).

Example 15 3β-Methoxy-17,17-ethylenedioxyandrost-5-ene-7β- and 7α-ol (22and 23)

3β-Methoxyandrost-5-ene-17-one (19) was ketalized using ethylene glycoland p-toluene sulfonic acid in toluene, heated to reflux for 7 h; yield95%, m.p. 138-40° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.35 (1H, d, J=4.88 Hz, 6-H), 3.9 (5H, m,O—CH₂—CH₂—O and 7α-H), 3.36 (3H, s, OCH₃), 3.05 (1H, m, 3α-H), 1.0 (3H,s, 19-CH₃), 0.86 (3H, s, 18-CH₃).

The ketalized produce was oxidized at the allylic 7 position, usingsodium periodate and t-butylhydroperoxide with sodium bicarbonate(Marwah and Lardy, U.S. Pat. No. 5,869,709 (1999), incorporated hereinby reference in its entirety,) to produce3β-methoxy-17,17-ethylenedioxyandrost-5-ene-7-one (21) in 62% yield;m.p. 190-192° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.7 (1H, d, J=1.96 Hz, 6-H), 3.89 (4H, m,—O—CH₂—CH₂—O—), 3.38 (3H, s, OCH₃), 3.2 (1H, m, 3α-H), 1.19 (3H, s,19-CH₃), 0.87 (3H, s, 18-CH₃).

Subsequent reduction of the carbonyl group at position 7 of product 21with sodium borohydride in a mixture of methylene chloride-methanol(1:9) at room temperature afforded 7-hydroxy derivatives. ¹H NMRspectrum of the product showed 7α- and 7β-forms in 2:8 ratio. Thediastereomers were separated by column chromatography on silica gel(eluent, ethyl acetate:hexane, 3:7).3β-Methoxy-17,17-ethylenedioxyandrost-5-ene-7β-ol (22) melted at 173-75°C.

¹H NMR (CDCl₃, 200 MHz): δ 5.29 (1H, t, J=1.95, 6-H), 3.89 (5H, m,O—CH₂—CH₂—O and 7α-H), 3.36 (3H, s, OCH₃), 3.14 (1H, m, 3α-H), 1.05 (3H,s, 19-CH₃), 0.88 (3H, s, 18-CH₃).

HRMS m/z: 362.2505 for C₂₂H₃₄O₄ requires 362.2457 (M⁺, 8.7%), 344.2338for C₂₂H₃₂O₃ requires 344.2351 (M⁺, —H₂O, 2.4%), 329.2170 for C₂₁H₂₉O₃requires 329.2117 (M⁺, H₂O, —CH₃, 7.2%), 261.1886 for C₁₇H₂₅O₂ requires261.1854 (M⁺, −101, 100%), 229.1628 for C₁₆H₂₁O requires 229.1592 (M⁺,−101, CH₃OH, 8.3%), 211 for C₁₆H₁₉ (M⁺, −101, CH₃OH, —H₂O, 2%).

3β-Methoxy-17,17-ethylenedioxyandrost-5-ene-7α-ol (23) melted at 183-84°C.

¹H NMR (CDCl₃, 200 MHz): δ 5.62 (1H, dd, J=1.96, 5.62 Hz, 6-H), 3.89(4H, m, O—CH₂—Ch₂-O), 3.89 (1H, 7β-H, merged in the ketal protons), 3.36(3H, s, OCH₃), 3.14 (1H, m, 3α-H), 0.99 (3H, s, 19-CH₃), 0.87 (3H, s,18-CH₃).

HRMS m/z: 362.2447 for C₂₂H₃₄O₄ requires 362.2457 (M⁺, 7%), 344.2347 forC₂₂H₃₂O₃ requires 344.2351 (M⁺, —H₂O, 3.5%), 330.2228 for C₂₁H₃₀O₃requires 330.2195 (M⁺, —CH₃OH, 1.6%), 329.2149 for C₂₁H₂₉O₃ requires329.2117 (M⁺, —H₂O, —CH₃, 4.2%), 261.1838 for C₁₇H₂₅O₂ requires 261.1854(M⁺, −101, 100%), 229.1594 for C₁₆H₂₁O requires 229.1592 (M⁺, −101,CH₃OH, 10%), 211 for C₁₆H₁₉ (M⁺, −101, CH₃OH, —H₂O, 2%).

Example 16

3β-Methoxyandrost-5-ene-7β,17β-diol (24). 24 was prepared by reductionof 3β-methoxyandrost-5-ene-7,17-dione 20, with sodium borohydride in amixture of methylene dichloride-methanol at room temperature. Theproduct was purified by column chromatography.

¹H NMR (CDCl₃, 200 MHz): δ 5.29 (1H, t, J=1.95, 6-H), 3.84 (1H, dt,J=8.3 Hz, 7α-H), 3.65 (1H, t, J=8.3 Hz, 17α-H), 3.36 (3H, s, OCH₃), 3.09(1H, m, 3α-H), 1.06 (3H, s, 19-CH₃), 0.78 (3H, s, 18-CH₃).

Example 17

3β-Acetoxy-7-methoxyandrost-5-ene-17-one (26). A mixture ofN-bromosuccimimide (2.15 g, 0.012 mol) and azobisisobutyronitrile (50mg), a radical initiator, was added in one lot to refluxing solution of3β-acetoxyandrost-5-ene-17-one (3.3 g, 0.01 mol) andazobisisobutyronitrile (50 mg) in carbon tetrachloride (70 mL). Themixture was refluxed for 20 min, cooled and solid succinimide wasremoved by filtration. The clear filtrate was concentrated at 20° C. andtriturated with petroleum ether, cooled at 0° C. to obtain the whitecrystalline 7α-bromo derivative 25; yield 2.8 g (68.6%).

¹H NMR (CDCl₃, 200 MHz): δ 5.78 (1H, d, J=5.13 Hz, 6-H), 4.76 (1H, m.7β-H), 4.68 (1H, m, 3α-H), 2.07 (3H, S, OCOCH₃), 1.07 (3H, s, 19-CH₃),0.91 (3H, s. 18-CH₃).

To a clear solution of 3β-acetoxy-7α-bromoandrost-5-ene-17-one, 25, (0.5g) in methanol (20 mL) was added silica gel (2.0 g, 70-230 mesh) and themixture was stirred for 2 h at room temperature. The methanolic solutionwas cooled, neutralized with saturated sodium bicarbonate solution, andfiltered on a thin bed of celite. The clear filtrate was cooled at 0° C.to obtain the white crystalline 7-methoxy derivative 26. Yield 0.32 g(72.7%), m.p. 175-78° C. The NMR spectrum of the white solid showed 55%7β-methoxy- and 45% 7α-methoxy-compound. Spectral data described belowwere deduced from the spectrum of the mixture. The biological activitywas assayed as the mixture (Table I).

7α-methoxy-derivative: ¹H NMR (CDCl₃, 200 MHz): δ 5.79 (1H, d, J=5.13Hz, 6-H), 4.65 (1H, m, 3α-H), 3.49 (1H, m, 7(3-H), 3.37 (3H, s,)CH₃),2.05 (3H, s, OCOCH₃), 1.03 (3H, s, 19-CH₃), 0.87 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 223.5 (C-17), 171 (C═O acetate), 146.5(C-5), 121.0 (C-6), 81.3 (C-3), 72.29 (C-7), 56.5 (OCH₃), 44.43, 42.86,37.03 (C-methines), 38.14, 36.62, 35.71, 30.93, 27.44, 21.83, 20.09(C-methylenes), 21.32 (OCOCH₃), 18.17 (C-19), 13.06 (C-18).

7α-methoxy-derivative: ¹H NMR (CDCl₃, 200 MHz): δ 5.53 (1H, s, 6-H),4.65 (1H, m, 3α-H), 3.5 (1H, m, 7α-H), 3.34 (3H, s, OCH₃), 2.05 (3H, s,OCOCH₃), 1.08 (3H, s, 19-CH₃), 0.89 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 223.5 (C-17), 171 (C═O acetate), 143.0(C-5), 121.8 (C-6), 81.3 (C-3), 73.24 (C-7), 55.0 (OCH₃), 51.33, 48.18,37.03 (C-methines), 37.83, 36.37, 35.92, 31.19, 27.6, 23.66, 20.39(C-methylenes), 21.32 (OCOCH₃), 18.95 (C-19), 13.55 (C-18).

LCMS (APES, positive): m/z 383.3 (M+Na, 100%), 359.2 (M, −1, 2.5%),299.3 (M, −60, 47%), 269.2 (M, −60, −32, 4%).

Example 17

3β-Methoxy-17β-hydroxyandrost-5-ene-7-one (28).3β-methoxyandrost-5-ene-17-one (19, 0.7 g, 2.3 mmol) was dissolved indry tetrahydrofuran (20 mL) and the solution was cooled to 0° C. Lithiumtri-t-butoxyaluminum hydride (1.2 g) was added to the cooled solutionand the mixture was stirred at the same temperature for 2.5 h. Excessbase was neutralized with dilute acetic acid and the produce wasextracted with methylene chloride. 3β-methoxyandrost-5-ene-17β-ol (27)was crystallized from methanol; yield 0.6 g (86%), m.p. 145-47° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.35 (1H, d, J=5.3 Hz, 6-H), 3.65 (1H, t,J=7.5 Hz, 17α-H), 3.36 (3H, s, OCH₃), 3.07 (1H, m, 3α-H), 1.02 (3H, s,19-CH₃), 0.76 (3H, s, 18-CH₃).

3β-methoxyandrost-5-ene-17β-ol (27) was subjected to air oxidation inthe presence of N-hydroxyphthalimide and the producT3β-methoxy-17β-hydroxyandrost-5-ene-7-one (28) was obtained in 53%yield. An analytical sample was obtained by recrystallization fromacetone-hexane, m.p. 202-4° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.7 (1H, d, J=1.47, 6-H), 3.36 (1H, t, J=7.8,17α-H), 3.38 (3H, s, OCH₃), 3.2 (1H, m, 3α-H), 1.21 (3H, s, 19-CH₃),0.77 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 202 (C-7), 165.9 (C-5), 125.8 (C-6), 80.8,78.9 (C-3, 17), 55.9, 50.03, 45.22, 44.9, 43.32, 38.72, 38.58, 36.22,35.63, 30.46, 27.56, 25.78, 20.85, 17.3, 11.1 (C-1, 2, 4, 8-16, 18, 19,OCH₃).

LCMS (API-ES, positive ion mode): λmax 241.5, m/z: 659.5 ((2M+Na)⁺,18%)), 341.2 ((M+Na)⁺, 100%)), 319.2 ((M+1)⁺, 22%)).

Example 18

3β-Methoxy-17β-acetoxyandrost-5-ene-7-one (29).3β-methoxy-17β-hydroxyandrost-5-ene-7-one (28) was acetylated inpyridine-acetic anhydride mixture at room temperature to obtain product29, m.p. 168-70° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.71 (1H, d, J=1.73 Hz, 6-H), 4.63 (1H, t,J=7.1 Hz, 17α-H), 3.41 (3H, s, OCH₃), 3.21 (1H, m. 3α-H), 2.08 (3H, s,OCOCH₃), 1.2 (3H, s, 19-CH₃), 0.81 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 201.3 (C-7), 171 (C═O acetate), 165.7 (C-5),125.8 (C-6), 81.9, 78.9 (C-3, 17), 55.9, 49.9, 44.9, 44.6 (C-8, 9, 14and OCH₃), 38.6, 36.2, 35.8, 30.46, 27.5, 25.8, 20.7 (C-1, 2, 4, 11, 12,15, 16), 21.1, 17.3, 12.0 (C-18, 19, OCOCH₃).

HRMS m/z: 360.2311 for C₂₂H₃₂O₄ requires 360.23 (M⁺, 100%), 329.2152 forC₂₁H₂₉O₃ requires 329.2117 (M⁺-CH₃, 42%), 328.2101 for C₂₁H₂₈O₃ requires328.2038 (M⁺-CHOH, 9.5%), 300.2061 for C2₂₀H2₂₈O₂ requires 300.2089(M⁺-CH₃COOH, 12%), 268.1840 for C₁₉H₂₄O requires 268.1827 (M⁺-CH₃OH,—CH₃COOH, 6%), 253.1640 for C₁₈H₂₁O requires 253.1592 (M⁺-CH₃OH,—CH₃COOH, —CH₃, 20%).

Example 19

3β-t-Butoxyandrost-5-ene-17-one (30). The product was prepared by theprocedure of Armstrong et al. (Tet. Lett., 29:2483-2486 (1988),incorporated herein by reference in its entirety), utilizing t-butyltrichloroacetimidate and borontrifluoro etherate in a solution ofdichloromethane and cyclohexane, in 67.2% yield; m.p. 185-87° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.34 (1H, d, J=4.89 Hz, 6-H), 3.32 (1H, m,3α-H), 1.2 (9H, s, t-Bu), 1.02 (3H, s, 19-CH₃), 0.89 (3H, s, 18-CH₃).

Example 20

3β-t-Butoxyandrost-5-ene-7,17-dione (31).3β-t-butoxyandrost-5-ene-17-one (30) was oxidized at the 7 position toobtain 3β-t-butoxyandrost-5-ene-7,17-dione (31) in 87% yield m.p.189-91° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.71 (1H, s, 6-H), 3.48 (1H, m, 3α-H), 1.2(9H, s, t-Bu), 1.2 (3H, s, 19-CH₃), 0.89 (3H, s, 18-CH₃).

HRMS m/z: 358.2502 for C₂₃H₃₄O₃ requires 358.2508 (M⁺, 2.4%), 302.1866for C₁₉H₂₆O₃ requires 302.1882 (M⁺-(CH₃)₂C═CH₂, 52%), 284.1798 forC₁₉H₂₄O₂ requires 284.1776 (M⁺-(CH₃)₂C═CH₂, —H₂O, 17%), 274.1965 forC₁₈H₂₆O₂ requires 274.1933 (M⁺-CO, —(CH₃)₂C═CH₂, 21%), 256.1843 forC₁₈H₂₄O requires 256.1827 (M+-H₂O, —CO, —(CH₃)₂C═CH₂, 25%), 246.1563 forC₁₆H₂₂O₂ requires 246.1620 (M⁺-CO, —CH₂═CH₂, —(CH₃)₂C═CH₂, 100%),231.1379 for C₁₅H₁₉O₂ requires 231.1385 (M⁺-CO, —CH₃, —CH₂═CH₂,—(CH₃)₂C═CH₂, 9%), 228.1551 for C₁₆H₂₀O requires 228.1514 (M⁺-CO,—CH₂═CH₂, —H₂O, —(CH₃)₂C═CH₂, 43%), 213.1383 for C₁₅H₁₇O requires231.1446 (M⁺-CO, —H₂O, —CH₂═CH₂, —CH₃, —(CH₃)₂C═CH₂, 8%).

Example 21 3β-t-Butyldimethylsilyloxyandrost-5-ene-7,17-dione (32)

3β-hydroxyandrost-5-ene-7,17-dione (2) (0.3 g, 0.99 mmol) was dissolvedin anhydrous dimethylformamide (10.0 mL). Imidazol (0.6 g, 8.82 mmol)and to-butyldimethysilyl chloride (0.3 g, 1.99 mmol) were added insequence and the mixture was stirred at room temperature under argon for3 h. The mixture was poured into cold water and the product wasextracted with ether and washed with dilute acetic acid, saturatedbicarbonate solution, then by water and brine. The solution was driedover MgSO₄ and the solvent was removed. The crude compound wascrystallized from aqueous methanol to afford white crystals of3β-t-butyldimethylsilyloxyandrost-5-ene-7,17-dione (32) in 92.6% yield(0.37 g), m.p. 135-6° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.73 (1H, s, 6-H), 3.61 (1H, m, 3α-H), 1.22(3H, s, 19-CH₃), 0.89 (12H, s, 18-CH₃ and C(CH₃)₃), 0.07 (6H, s,Si(CH₃)₂).

¹³C NMR (CDCl₃, 300 MHz): δ 220.5 (C-17), 201 (C-7), 166.8 (C-5), 125.6(C-6), 71.1 (C-3), 50.15, 46.0, 44.33 (C-8, 9, 14), 48.0, 38.5, 18.2(C-18,19,0-Me₃); 42.62, 36.35, 35.63, 31.67, 30.73, 24.17, 20.57 (C-1,2, 4, 11, 12, 15, 16), 25.82 ((CH₃)₃), 17.41, 13.74, (C-18, 19), −4.0(Si(CH₃)₂.

LCMS (AP, positive ion mode): λmax 240, m/z 417.3 ((M+1)⁺, 71%), 285.2(M+1, -TBDMSiOH, 100%), 267.2 (M+1, -TBDMSiOH, —H₂O, 14%), 249.3 (M+1,-TBDMSiOH, -2(H₂O), 1%), 243.2 (M+1, —CH₂═C═O, 6%).

Example 22

3β,17β-di(t-butyldimethylsilyloxy)androst-5-ene-7-one (33). The productwas prepared from 3β-17β-dihydroxyandrost-5-ene-7-one as described forcompound 32. M.P. 99-101° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.65 (1H, s, 6-H), 3.60 (2H, m. 3α- and17α-H), 1.18 (3H, s, 19-CH₃), 0.74 (3H, s, 18-CH₃), 0.87 (18H, s,Si-t-butyl), 0.04 (12H, s, Si-Me₂).

Example 23

3β-Acetoxyandrost-5-ene-7β,17β-di(t-butyldimethylsilyl)ether (35).Prepared from 3β-acetoxyandrost-5-ene-7β,17β-diol (34) andt-butyldimethylsilyl chloride in dichloromethane. M.p. 85-87° C.

LCMS (APCl, positive ion mode): m/z 575.25 (M−1)⁺, 515.35 (M−1,—CH₃COOH, 40%), 385.25 (M+1, —CH₃COOH, -TBDMSiOH, 11%), 253.15 (M+1,—CH₃COOH, -2(TBDMSiOH), 29%), 238.15 (M+1, —CH₃COOH, -2(TBDMSiOH), —CH₃,7%).

Example 24

3β-Acetoxy-17β-t-butyldimethylsilyloxyandrost-5-ene-7-one (36). Preparedfrom 3β-acetoxy-17β-hydroxyandrost-5-ene-7-one (4). M.p. 202-4° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.7 (1H, d, J=1.3 Hz, 6-H), 4.72 (1H, m,3α-H), 3.56 (1H, dd, J=1.7 Hz, 17α-H), 2.07 (3H, s, OCOCH₃), 1.22 (3H,s, 19-CH₃), 0.88 (9H, s, Si-t-butyl), 0.72 (3H, s, 18-CH₃), 0.009 (6H,s. SiMe₂).

¹³C NMR (CDCl₃, 300 MHz): δ 201.5 (C-7), 170 (OCOCH₃), 164.1 (C-5),126.5 (C-6), 80.9 (C-17), 72.1 (C-3), 50.1, 45.3, 44.5 (C-methines),37.8, 36.0, 35.9, 30.9, 27.3, 26.2, 20.87 (C-methylenes), 25.9, 25.82,21.2, 17.3, 11.3, −4.5, −4.9 (C-Methyls).

Example 25 3β-(2-tetrahydropyranoxy)androst-5-ene-7,17-dione (37)

3β-hydroxyandrost-5-ene-7,17-dione (2) (0.5 g, 1.65 mmol) was placed ina dried, argon-flushed flask where it was dissolved in drydichloromethane (20.0 mL). Pyridinium toluene-p-sulfonate (0.043 g. 0.17mmol) was added followed by 3,4-dihydro-2H-pyran (0.23 mL, 2.5 mmol) andthe solution was stirred at room temperature, under an atmosphere ofargon, for 2-3 h. The reaction was quenched by adding water and theproduct was extracted thrice with ether (3×20 mL). The combined etherextracts were washed with dilute acetic acid, water and bicarbonatesolution and dried (MgSO₄). Solvent was removed and the crude productwas purified using chromatography on silica gel (ethyl acetate-hexane,15:85, v/v). The compound first eluted was identified asandrosta-3,5-diene-7,17-dione (0.150 g), m.p. 167-8° C.

Further elution with the same solvent gave3β-(2-tetrahydropyranoxy)androst-5-ene-7,17-dione (37) as a white solid,which was crystallized from methanol (0.43 g, 68%), m.p. 137-39° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.76, 5.73* (1H, dd, J=2 Hz, 6-H), 4.7 (1H,m, 2-H Pyran), 3.91 (1H, m, 6-H pyran), 3.67 (1H, m, 3α-H), 3.51 (1H, m,6-H pyran), 1.23 (3H, s, 19-CH₃), 0.90 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 221 (C-17), 201 (C-7), 167, 166.4* (C-5),125.8 (C-6), 97.4, 97.1*, 74.7, 74.53* (C-3, C-2 (Pyran)), 62.86 (C-6(Pyran)), 50.05, 45.69, 44.26 (C-8, 9, 14), 47.82, 38.62 (C-10, 13),40.10, 38.80, 36.41, 36.15, 35.59, 31.07, 30.68, 29.31, 27.64, 25.35,24.14, 20.52, 19.83 (C-1, 2, 4, 11, 12, 15, 16, C-3, 4, 5 (Pyran))*,17.35, 13.71 (C-18, 19).

LCMS (APES, positive): λmax 238; m/z 795.5 (2M+Na, 100%), 409.1 (M+Na,85%), 285.1 (M, −101, 14%).

* Extra peaks are due to the diastereomers.

Example 26 3β-dodecanoxyandrost-5-ene-7,17-dione (39)

A solution of DHEA (1) (2.88 g. 0.01 mol) in anhydrous tetrahydrofuran(20 mL) was added to a mixture of sodium hydride (0.5 g, 60% in oil,washed twice with pentane) in anhydrous tetrahydrofuran (10 mL). Themixture was refluxed for 2 h, cooled to room temperature, and a solutionof 1-bromododecane (2.74 g, 0.11 mol) was added slowly (10 min). Thesolution was refluxed for 4 h, cooled and poured into ice water and theproduct was extracted with ether-ethyl acetate mixture (1:1).3β-Dodecanoxyandrost-5-ene-17-one (38) was purified by columnchromatography on silica gel (eluent ethyl acetate-petroleum ether,1:9), yield 0.6 g (43%, based on 30.5% conversion). Further elution withthe same solvents at (4:6) afforded unreacted DHEA (2.0 g, 69.5%).

¹H NMR (CDCl₃, 200 MHz): δ 5.38 (1H, d, J=5.12 Hz, 6-H), 3.45 (2H, t,J=6.6, 6.83 Hz, —CH₂—O), 3.14 (1H, m. 3α-H), 1.03 (3H, s, 19-CH₃), 0.89(3H, s, 18-CH₃).

3β-dodecanoxyandrost-5-ene-17-one (38) was subjected to oxidation by aprocedure to be published and the product,3β-dodecanoxyandrost-5-ene-7,17-dione (39) was obtained in 82.8% yield,m.p. 70° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.74 (1H, d, J=1.71 Hz, 6-H), 3.47 (2H, t,J=6.35, 6.83 Hz, —CH₂—O), 3.27 (1H, m, 3α-H), 1.22 (3H, s, 19-CH₃), 0.9(3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 220 (C-17), 200 (C-7), 166.0 (C-5), 125.8(C-6), 77.45 (C-3), 68.58 (—CH₂—O), 50.14, 45.75, 44.3 (C-methines),39.19, 36.31, 35.58, 31.9, 30.7, 30.1, 29.59, 29.46, 29.34, 28.07,26.16, 24.15, 22.66, 20.55 (C-methylenes), 17.35, 14.07, 13.7(C-methyls).

HRMS m/z: 470.3758 for C₃₁H₅₀O₃ requires 470.3760 (M⁺, 23%), 301.1783for C₁₉H₂₅O₃ requires 301.1804 (M⁺-C₁₂H₂₅, 10%), 285.1870 for C₁₉H₂₅O₂requires 285.1854 (M⁺—C₁₂H₁₅O, 12%), 284.1610 for C₁₉H₂₄O₂ requires284.1776 (M⁺-C₁₂H₁₅OH, 5.6%).

Example 27 3β-(1′-ethoxy)ethoxyandrost-5-ene-7,17-dione (41)

To a solution of DHEA (1) (1.5 g, 0.005 mol) and p-toluene sulfonic acid(35 mg) in methylene dichloride (20 mL), a solution of ethyl vinyl ether(1.17 g, 0.016 mol) in methylene dichloride was added slowly during 2 hat room temperature. The solution was stirred for an additional 6 h andpoured into cold water. The organic layer was separated and the productwas subject to column chromatography on silica gel (eluent, ethylacetate-petroleum ether, 1:9) to afford3β-(1′-ethoxy)ethoxyandrost-5-ene-17-one (40) as a viscous mass (0.92 g,84% based on 55% conversion).

¹H NMR (CDCl₃, 200 MHz): δ 5.37 (1H, s, 6-H), 4.78 (1H, q, J=5.37 Hz,O—CH—O), 3.68 (2H, m, —CH₂—O), 3.5 (1H, m, 3α-H), 1.31 (3H, d, J=5.13,CH₃), 1.2 (3H, m, CH₃), 1.03 (3H, s, 19-CH₃), 0.89 (3H, s, 18-CH₃).

3β-(1′-ethoxy)ethoxyandrost-5-ene-17-one was oxidized at the allylic 7position of the steroid using air and N-hydroxy phthalimide as describedbefore to obtain 3β-(1′-ethoxy)ethoxyandrost-5-ene-7,17-dione (41) in50% yield.

¹H NMR (CDCl₃, 200 MHz): δ 5.74 (1H, s, 6-H), 4.79 (1H, m. O—CH—O), 3.58(3H, m, —CH₂—O and 3α-H), 1.34 (3H, d, J=5.13 Hz, CH₃), 1.22 (3H, m,CH₃), 1.22 (3H, s, 19-CH₃), 0.9 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 220 (C-17), 200 (C-7), 166.0 (C-5), 125.77(C-6), 98.42 (O—CH—O), 73.89 (C-3), 60.19 (O—CH₂), 50.06, 45.69, 44.27(C-methines), 41.88, 40.03, 35.58, 30.67, 29.1, 24.1, 20.5(C-methylenes), 20.7, 17.34, 15.28, 13.71 (C-methyls).

Carbonate Derivatives.

Example 28 3β-Carbomethoxyandrost-5-ene-7,17-dione (42)

3β-Hydroxyandrost-5-ene-7,17-dione (2) (0.5 g, 0.0016 mol) was dissolvedin dry pyridine (6 mL) and the solution was cooled to 0-5° C. Methylchlorofmate (0.19 g, 0.002 mol) was added dropwise during 15 min and themixture was stirred at the same temperature for 3 h. The reactionmixture was quenched with water and the product was extracted withmethylene dichloride. The organic layer was washed, dried and solventwas removed. The crude product was purified by column chromatography onsilica gel (eluent: acetone-hexane, 2:8) to afford 0.42 g (89%,conversion 80%) of white solid which was crystallized from methanol,m.p. 168-70° C.

¹H NMR (CDCL₃), 200 MHz): δ 5.77 (1H, d, J=0.7 Hz, 6-H), 3.78 (3H, s,OCH₃), 4.62 (1H, m, 3α-H), 1.24 (3H, s, 19-CH₃), 0.9 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 220.1 (C-17), 200.2 (C-7), 164 (C-5), 155(O—C—O), 126.7 (C-6), 75.7 (C-3), 54.8 (OCH₃), 49.9, 45.7, 44.3, (CH—,8, 9 and 14), 37.7, 35.8, 35.6, 30.7, 27.2, 24.1, 20.6 (CH₂—, 1, 2, 4,11, 12, 15 and 16), 17.4, 13.7 (CH₃-18, and 19).

HRMS m/z: 360.1929 for C₂₁H₂₈O₅ requires 360.1937 (M⁺, 1.7%), 284.1789for C₁₉H₂₄O₂ requires 284.1776 CH₃OH, —CO₂, 100%), 269.1521 for C₁₈H₂₁O₂requires 269.1541 (M⁺-CH₃OH, —CO₂, —CH₃, 54.%), 256.1856 for C₁₈H₂₄Orequires 256.1827 (M⁺-CH₃OH, —CO₂, —CO, 25.6%), 241.1623 for C₁₇H₂₁Orequires 241.1592 (M⁺-CH₃OH, —CO₂, —CH₃, —CO, 11%), 277.1461 for C₁₆H₁₉Orequires 227.1463 (M⁺-CH₃OH, —CO₂, —CH₃,—CO, —CH₂, 5.6%).

Example 29 3β-Carboallyloxyandrost-5-ene-7,17-dione (43)

Compound 43 was prepared from 7-oxo-DHEA (2) (1.0 mmol) andallylchloroformate (4.0 mmol) in tetrahydrofuran-pyridine mixture at0-5° C. After stirring 6 hours only 30% conversion had occurred; fromthe mixture the product was isolated in 78% yield (based on 30%conversion) by column chromatography. M.p. 159-61° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.92 (1H, m, ═CH₂—), 5.74 (1H, d, J=1.7 Hz,6-H), 5.38 (q), 5.29 (m), 5.23 (m) (2H, J=18.5, 11.6, 1.47 Hz, —CH₂═),4.62 (2H, dt, J=5.86, 1.22, 1.47 Hz, —CH₂—O—), 4.62 (1H, m, 3α-H) 1.21(3H, s, 19-CH₃), 0.87 (3H, s, 18-CH₃).

¹³C NMR (DCCl₃, 300 MHz): δ 219.2 (C-17), 200 (c-7), 164 (c-5), 154(O—C—O), 131.5 (═O—), 126.5 (C-6), 118.7 (—OH₂═), 75.7 (C-3), 68.24(O—CH₂), 50, 45.8, 44.3, (CH—, 8, 9 and 14), 47.64, 38.33 (c-, 10, 13),37.7, 35.8, 35.4, 30.7, 27.2, 24.02, 20.53, (CH₂—, 1, 2, 4, 11, 12, 15,and 16), 17.3, 13.7 (CH₃-18, and 19).

LCMS (APES, positive): Amax 234 nm; λmax 409.1 (M+Na, 100%), 307.1 (M,−102, +Na, 80%), 285 (M, −101, 52%), 256 (M, −102, −28).

Example 30

3β-Carboethoxyandrost-5-ene-7,17-dione (44). Prepared from (2) andethylchloroformate in pyridine at 0-5° C. Yield 90%, m.p. 187-8° C.

¹H NMR (CDCl₃, 300 MHz): δ 5.77 (1H, d, J=1.65 Hz, 6-H), 4.6 (1H, m,3α-H), 4.2 (2H, q, J=7.2 Hz, —CH₂—O), 1.32 (3H, t, J=7.2 Hz, CH₃), 1.23(3H, s, 19-CH₃), 0.9 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 220.2 (C-17), 200.7 (C-7), 164.4 (C-5),154.3 (O—C—O), 126.7 (C-6), 75.4 (C-3), 64.1 (O—CH₂), 49.9, 45.7, 44.3,(CH—, 8, 9 and 14), 47.8, 38.4 (C—, 10, 13), 37.7, 35.8, 35.6, 30.7,27.2, 24.1, 20.5 (CH₂—, 1, 2, 4, 11, 12, 15, and 16), 14.4, 14.2, 13.8(CH₃, CH₃-18, 19).

Example 31

3β-Carboisobutoxyandrost-5-ene-7,17-dione (45). Prepared from (2) andisobutylchloroformate in pyridine at 0-5° C. Yield 78%, m.p. 132-3° C.

¹H NMR (CDCl₃, 300 (MHz): δ 5.77 (1H, d, J=1.66 Hz, 6-H), 4.6 (1H, m,3α-H), 3.92 (2H, d, J=6.8 Hz, —CH₂—O), 1.24 (3H, s, 19-CH₃), 0.96 (6H,d, J=6.6 Hz, (CH₃)₂), 0.9 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 220.2 (C-17), 200.7 (C-7), 164.4 (C-5),154.5 (O—C—O), 126.6 (C-6), 75.5 (C-3), 74.14 (O—CH₂), 49.9, 45.7, 44.3,(CH—, 8, 9, and 14), 27.7 (—CH<), 37.8, 35.8, 35.6, 30.7, 27.2, 24.1,20.6 (CH₂—, 1, 2, 4, 11, 12, 15, and 16), 18.91 (CH₃), 18.87 (CH₃),17.35 (19-CH₃), 13.7 (18-CH₃).

Example 32 3β,17β-dicarbomethoxyandrost-5-ene-7-one (46). Prepared from3β,17β-dihydroxyandrost-5-ene-7-one and methylchloroformate in pyridineat 0-5° C. Yield 81%, m.p. 167-9° C.

¹H NMR (CDCl₃, 300 MHz): δ 5.73 (1H, d, J=1.66 Hz, 6-H), 4.57 (2H, m,3α-H, 17α-H), 3.79 (3H, s, CH₃O), 3.77 (3H, s, CH₃O), 1.2 (3H, s,19-CH₃), 0.84 (3H, s, 18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 200.97 (C-7), 163.89 (c-5), 155.7, 154.88(O—C—O), 126.6 (C-6), 85.65 (C-17), 75.8 (C-3),54.72, 54.58)OCH₃),49.65, 44.91, 44.55, (CH—, 8, 9 and 14), 43.03, 38.32 (C—, 10, 13),37.7, 35.82, 35.65, 27.43, 27.21, 25.68, 20.67 (CH₂—, 1, 2, 4, 11, 12,15, and 16), 11.94 (CH₃-18, and 19).

Example 33

3β-Carbooctyloxyandrost-5-ene-7,17-dione (47). Prepared from (2) andoctylchloroformate in pyridine at 0-5° C. The product was purified bycolumn chromatography on silica gel. Yield 65%, m.p. 72-3° C.

¹H NMR (CDCl₃, 300 MHz): δ 5.77 (1H, d, J=1.66 Hz), 4.6 (1H, m, 3α-H),4.13 (2H, t, J=6.6 Hz, —CH₂—O), 1.23 (3H, s, 19-CH₃), 0.88 (3H, s,18-CH₃).

¹³C NMR (CDCl₃, 300 MHz): δ 219.76 (C-17), 200.4 (C-7), 164.28 (C-5),154.28 (O—C—O), 126.53 (C-6), 75.34 (C-3), 68.08 (O—CH₂), 49.81, 45.6,44.2, (CH—, 8, 9 and 14), 47.69 (c-13), 38.31 (c-10), 37.67, 35.74,35.46, 31.65, 30.61, 29.06, 29.04, 28.54, 27.15, 25.6, 24.05, 22.52,20.47 (methylenes), 14.02, (CH₃), 17.25 (19-CH₃), 13.65 (18-CH₃).

LCMS (APES, positive): m/z 459.2 (M+1, 8%), 285.1 (M, −174, 100%).

Example 34 3β-Carbo(9-fluorenyl)methoxyandrost-5-ene-7,17-dione (48)

A mixture of 7-oxoDHEA (2) (0.3 g, mmol) and9-fluorenylmethylchloroformate (0.285 g, 1.1 mmol) in pyridine (4.0 mL)was stirred at room temperature for 1 hour. The product was purified bycolumn chromatography on silica gel, to yield 57% (0.3 g), m.p. 98-103°C., λ_(max) 260 nm.

¹H NMR (CDCl₃, 200 MHz): δ 7.77 (2H, d, J=7.11 Hz, Ar—H), 7.62 (2H, d,J=6.83 Hz, Ar—H), 7.37 (4H, m, Ar—H), 5.77 (1H, d, J=1.46 Hz, 6-H), 4.61(1H, m, 3-H), 4.43 (2H, d, J=6.96 Hz, AB system, OCH₂), 4.26 (1H, t,J=7.08 Hz, AB system, Ar—H), 1.25 (3H, s, 19-CH₃), 0.90 (3H, s, 18-CH₃).

LC-MS (API-ES) m/z: 547 (M+23, 100%), 285 (M+1, −240, 3%).

Example 35

3β-Carbomethoxyandrost-5-ene-7,17β-diol (49).3β-Carbomethoxyandrost-5-ene-7,17-dione (42) (0.5 g, 0.0014 mol) wasdissolved in a mixture of dichloromethane (5 mL) and methanol (10 mL).The mixture was stirred at room temperature and finely powdered sodiumborohydride (0.16 g, 0.004 mol) was added slowly. After 15 min thereaction mixture was quenched with water and the product was extractedwith methylene dichloride. The organic layer was washed, dried andsolvent removed. The product was crystallized from acetone-petroleumether to afford 0.35 g (70%) of 3β-carbomethoxyandrost-5-ene-7,17β-diol(49) as a white crystalline solid, m.p. 150-52° C. LC-MS and ¹H NMRanalysis of the product showed a 8:2 ratio of isomeric 7β-hydroxy and7α-hydroxy compound.

¹H NMR for 7β-hydroxy isomer (CDCl₃, 200 MHz): δ 5.33 (1H, s, 6-H), 4.49(1H, m, 3α-H), 3.77 (3H, s, OCH₃), 3.84 (1H, d, J=7.08 Hz, 7α-H), 3.64(1H, s, 6-H), 4.49 (1H, m, 3α-H), 3.77 (3H, s, OCH₃), 3.84 (1H, d,J=7.08 Hz, 7α-H), 3.64 (1H, t, J=8.05 Hz, 17α-H), 1.07 (3H, s, 19-CH₃),0.77 (3H, s, 18-CH₃).

7α-hydroxy isomer showed different chemical shifts for 6 and 19 protons,deduced from the spectrum of the mixture (1H, d, J=4.89 Hz, 6-H), 1.02(3H, s, 19-CH₃).

LC-MS (API-ES) m/z: 387 (M+23 100%), 311 (M+23, −76, 25%), 271 (M+1,−76, −18, 5%), 253 (M+1, −76, −18, −18, 3%).

Example 36

3β-Carboethoxyandrost-5-ene-7β,17β-diol (50).3β-Carbomethoxyandrost-5-ene-7,17-dione (44) (0.3 g, 0.8 mmol) in 5 mLdichloromethane and 10 mL methanol was treated with cerium (III)chloride heptahydrate (0.3 g, 0.8 mmol). To this cold solution, finelypowdered sodium borohydride (0.09 g, 2.4 mmol) was added slowly. After15 minutes the solvent was evaporated to complete dryness, the solidcrude was taken up in dichloromethane, stirred for 30 minutes andfiltered on a small bed of celite. The organic layer was washed oncewith water, dried and the solvent was removed. The product wascrystallized from acetone-petroleum ether to afford 0.22 g (73%) of3β-carboethoxyandrost-5-ene-7β,17β-diol (50) as a white solid, m.p.170-71° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.33 (1H, s, 6-H), 4.48 (1H, m, 3α-H), 4.23(2H, q, J=7.08, OCH₂), 3.84 (1H, d, J=7.57 Hz, 7α-H), 3.64 (1H, t, J=8.3Hz, 17α-H), 1.31 (3H, t, J=7.08, CH₃), 1.07 (3H, s, 19-CH₃), 0.77 (3H,s, 18-CH₃).

LC-MS (API-ES) m/z: 401 (M+23, 100%), 311 (M+23, −90, 15%), 271 (M+1,−90, −18, 3%), 253 (M+1, −90, 18, −18, 5%).

Example 37

3β-Carbooctyloxyandrost-5-ene-7β,17β-diol (51).3β-Carbooctyloxyandrost-5-ene-7,17-dione (47) (0.3 g, 0.65 mmol) wasconverted to 51 as described for compound 49. The product wascrystallized from petroleum ether to afford 0.23 g (77%) of3β-carbooctyloxyandrost-5-ene-7β,17β-diol (51) as a white solid, m.p.86-87° C.

¹H NMR (CDCl₃, 200 MHz): δ 5.33 (1H, s, 6-H), 4.48 (1H, m, 3α-H), 4.12(2H, t, J=6.6, OCH₂), 3.89 (1H, d, J=7.54 Hz, 7α-H), 3.65 (1H, t, J=8.3Hx, 17α-H), 0.88 (3H, t, CH₃), 1.07 (3H, s, 19-CH₃), 0.77 (3H, s,18-CH₃₃).

LC-MS (API-ES) m/z: 485 (M+23, 100%), 311 (M+23, −174, 3%), 293 (M+23,−174, −18, 2%).

Induction of the Synthesis of Mitochondrial GlycerophosphateDehydrogenase and Cytosolic Malic Enzymes in Rat Livers

Enzymes GDPH Malic Conc. & % of # R b/a b′/a′ in diet control * AcO O O0.040 336 338 9 Glu-ester O O 0.085 166 306 10 Glu-triAc-ester O O 0.107164 234 12 AcO H/H Glu-ester 0.083 128 229 13 AcO O Glu-ester 0.093 204363 16 AcO Glu-ester/H AcO 0.071 241 367 17 AcO H/Glu-triAc- O 0.046 8193 ester 18 OTs H/H O 19 MeO H/H O 0.085 241 312 20 MeO O O 0.055 323424 21 MeO O O—CH₂—CH₂—O 0.042 194 311 22 MeO OH/H O—CH₂—CH₂—O 0.063 395595 23 MeO H/OH O—CH₂—CH₂—O 0.063 262 324 24 MeO OH/H OH/H 0.055 403 44425 AcO H/Br O 26 AcO MeO•H** O 0.064 328 459 27 MeO H/H OH/H 28 MeO OOH/H 0.055 322 307 29 MeO O AcO/H 0.06 188 166 30 tert-BuO H/H O 31tert-BuO O O 0.04 93 116 32 TBDMSO O O 0.036 241 313 33 TBDMSO OTBDMSO/H 0.085 168 380 34 AcO OH/H OH/H 0.046 497 508 35 AcO TBDMSO/HTBDMSO/H 0.082 123 100 36 AcO O TBDMSO/H 0.07 375 382 37 TetrahydropyranO O 0.064 280 357 38 Dodecanoxy H/H O 39 Dodecanoxy O O 0.068 156 189 40(Ethoxy)ethyl H/H O 41 (Ethoxy)ethyl O O 0.05 168 275 42 Carbomethoxy OO 0.1 500 786 43 Carboallyloxy O O 0.067 440 384 44 Carboethoxy O O0.045 265 373 45 Carbo, iso-butyloxy O O 0.048 253 515 46 Carbomethoxy OCarbomethoxy/H 0.051 295 292 47 Carbooctyloxy O O 0.055 256 312 48Carbo(9-fluorene) O O 0.09 218 310 methoxy 49 Carbomethoxy OH, H** OH/H0.042 377 359 50 Carboethoxy OH/H OH/H 0.044 277 321 51 CarbooctyloxyOH/H OH/H 0.054 343 260 Each compound was tested in a group of two orthree rats. Enzyme activity in the livers of rats fed the stock dietwithout supplementation is termed 100%. The abbreviation TBDMSO =tert-butyldimethylsilyl oxy. *Mean values from ten experiments (20 rats)in which 7-oxo-DHEA was the comparative standard. **Products tested asdiestereomeric mixture (% ratio of β and α, 26, 55:45, 49, 80:20) Thefollowing compounds did not induce the liver enzymes:17-oxoA-3β-glucopyranoside Me ester (8),3β-acetoxy-17-oxoA-7-glucopyranoside Me ester (17),3β-tert-butoxyandrost-5- ene-17-one (30),3β-acetoxyandrost-5-ene-7β,17β-di-tert-butyldimethylsilyl ether (35); A= androst-5-ene.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,some of which are disclosed herein. It will be apparent to the artisanthat other embodiments exist and do not depart from the scope of theinvention. Thus, the described embodiments are illustrative and shouldnot be construed as restrictive.

For any of the uses of formula 1 compounds described herein, e.g., inany of the examples above, the results or biological effects that areobtained using individual formula 1 compounds are optionally compared tothe results or biological effects that are obtained using a referenceformula 1 compound such as AET, AED, BrEA, positive controls or negativecontrols or to other known modulators of the biological activity,symptom or clinical condition of interest. Such comparisons provideguidance for using the formula 1 compounds in the different methods orclinical conditions. Such comparison information allows, e.g., tailoringof dosages and dosing schedules, routes of administration or the likefor individual applications for the formula 1 compounds.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any of the various specific embodiments,compounds or compositions described herein may be modified toincorporate other appropriate features, e.g., as shown in any other ofthe specific embodiments disclosed herein or in the cited references.

1. A compound having the structure

wherein the dotted line is an optional double bond; R¹, R², and R⁴independently are —OH, an ester or an ether; R³ is —OH, an ester, anether or halogen; R⁵ is —CH₃ or —CH₂OH; R⁶ is —H or —CH₃; and R⁷ is —S—.2. The compound of claim 1 wherein R¹, R² and R⁴ are —OH or an ester andR³ is —OH, an ester or —Br.
 3. The compound of claim 2 wherein R⁵ is—CH₃.
 4. The compound of claim 1 wherein the compound has the formula


5. The compound of claim 4 wherein R¹, R² and R⁴ are —OH or an ester andR³ is —OH, an ester or —Br.
 6. The compound of claim 5 wherein R⁵ and R⁶are —CH₃.
 7. The compound of claim 1 wherein the compound has thestructure


8. The compound of claim 1 wherein the compound has the formula


9. The compound of claim 8 wherein R¹, R² and R⁴ are —OH or an ester andR³ is —OH, an ester or —Br.
 10. The compound of claim 9 wherein R⁵ andR⁶ are —CH₃.
 11. The compound of claim 10 wherein the compound has thestructure


12. The compound of claim 11 wherein the compound has the structure


13. A pharmaceutical formulation comprising one or more excipients and acompound having the structure

wherein the dotted line is an optional double bond; R¹, R², and R⁴independently are —OH, an ester or an ether; R³ is —OH, an ester, anether or halogen; R⁵ is —CH₃ or —CH₂OH; R⁶ is —H or —CH₃; and R⁷ is —S—.14. The pharmaceutical formulation of claim 13 wherein R¹, R² and R⁴ are—OH or an ester, R³ is —OH, an ester or —Br and R⁵ and R⁶ are —CH₃. 15.The pharmaceutical formulation of claim 13 wherein the pharmaceuticalformulation has the formula


16. The pharmaceutical formulation of claim 16 wherein R¹, R² and R⁴ are—OH or an ester and R⁵ and R⁶ are —CH₃.
 17. The pharmaceuticalformulation of claim 13 wherein the pharmaceutical formulation has thestructure


18. The pharmaceutical formulation of claim 13 wherein the compound hasthe structure


19. The pharmaceutical formulation of claim 18 wherein the compound hasthe structure